Target-dependent reactions using structure-bridging oligonucleotides

ABSTRACT

The present invention relates to methods and compns. for treating nucleic acids, and in particular, methods and compns. for the detection and characterization of nucleic acid sequences and sequence changes. The invention provides methods for examg. the conformations assumed by single strands of nucleic acid, forming the basis of novel methods of detection of specific nucleic acid sequences. The present invention contemplates use of novel detection methods for, among other uses, clinical diagnostic purposes, including but not limited to the detection and identification of pathogenic organisms. Examples are presented for the analysis of  Mycobacterium tuberculosis  and hepatitis C virus genes.

This application is a 371 of PCT/US98/03194 filed May 5, 1998 which is aCIP of Ser. No. 08/851,588 filed May. 5, 1997, now U.S. Pat. No.6,214,545, and a CIP of Ser. No. 08/934,097 filed Sep. 19, 1997, nowU.S. Pat. No. 6,210,880, and a CIP of Ser. No. 08/034,205 filed Mar. 3,1998 and now U.S. Pat. No. 6,194,149.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for analyzingnucleic acids, and in particular, methods and compositions for detectionand characterization of nucleic acid sequences and sequence changes.

BACKGROUND OF THE INVENTION

The detection and characterization of specific nucleic acid sequencesand sequence changes have been utilized to detect the presence of viralor bacterial nucleic acid sequences indicative of an infection, thepresence of variants or alleles of mammalian genes associated withdisease and cancers, and the identification of the source of nucleicacids found in forensic samples, as well as in paternity determinations.As nucleic acid sequence data for genes from humans and pathogenicorganisms accumulates, the demand for fast, cost-effective, andeasy-to-use tests for as yet unknown, as well as known, mutations withinspecific sequences is rapidly increasing.

A handful of methods have been devised to scan nucleic acid segments formutations. One option is to determine the entire gene sequence of eachtest sample (e.g., a clinical sample suspected of containing bacterialstrain). For sequences under approximately 600 nucleotides, this may beaccomplished using amplified material (e.g., PCR reaction products).This avoids the time and expense associated with cloning the segment ofinterest. However, specialized equipment and highly trained personnelare required for DNA sequencing, and the method is too labor-intense andexpensive to be practical and effective in the clinical setting.

In view of the difficulties associated with sequencing, a given segmentof nucleic acid may be characterized on several other levels. At thelowest resolution, the size of the molecule can be determined byclectrophoresis by comparison to a known standard run on the same gel. Amore detailed picture of the molecule may be achieved by cleavage withcombinations of restriction enzymes prior to electrophoresis, to allowconstruction of an ordered map. The presence of specific sequenceswithin the fragment can be detected by hybridization of a labeled probe,or, as noted above, the precise nucleotide sequence can be determined bypartial chemical degradation or by primer extension in the presence ofchain-terminating nucleotide analogs.

For detection of single-base differences between like sequences (e.g.the wild type and a mutant form of a gene), the requirements of theanalysis are often at the highest level of resolution. For cases inwhich the position of the nucleotide in question is known in advance,several methods have been developed for examining single base changeswithout direct sequencing. For example, if a mutation of interesthappens to fall within a restriction recognition sequence, a change inthe pattern of digestion can be used as a diagnostic tool (e.g.,restriction fragment length polymorphism [RFLP] analysis). In this way,single point mutations can be detected by the creation or destruction ofRFLPs.

Single-base mutations have also been identified by cleavage of RNA-RNAor RNA-DNA heteroduplexes using RNaseA (Myers et al., Science 230:1242[1985] and Winter et al., Proc. Natl. Acad. Sci. USA 82:7575 [1985]).Mutations are detected and localized by the presence and size of the RNAfragments generated by cleavage at the mismatches. Single nucleotidemismatches in DNA heteroduplexes are also recognized and cleaved by somechemicals, providing an alternative strategy to detect single basesubstitutions, generically named the “Mismatch Chemical Cleavage” (MCC)(Gogos et al., Nucl. Acids Res., 18:6807-6817 [1990]). However, thismethod requires the use of osmium tetroxide and piperidine, two highlynoxious chemicals which are not suited for use in a clinical laboratory.Enzymes such as the bacteriophage T4 endonuclease VII have been used in“Enzymatic Mismatch Cleavage: (EMC) (Youil, et al., Genomics, 32:431[1996]). However, all of the mismatch cleavage methods lack sensitivityto some mismatch pairs, and all are prone to background cleavage atsites removed from the mismatch. Furthermore, the generation of purifiedfragments to be used in heteroduplex formation is both labor intensiveand time consuming.

RFLP analysis suffers from low sensitivity and requires a large amountof sample. When RFLP analysis is used for the detection of pointmutations, it is, by its nature, limited to the detection of only thosesingle base changes which fall within a restriction sequence of a knownrestriction endonuclease. Moreover, the majority of the availableenzymes have 4 to 6 base-pair recognition sequences, and cleave toofrequently for many large-scale DNA manipulations (Eckstein and Lilley(eds.), Nucleic Acids and Molecular Biology, vol. 2, Springer-Verlag,Heidelberg [1988]). Thus, it is applicable only in a small fraction ofcases, as most mutations do not fall within such sites.

A handful of rare-cutting restriction enzymes with 8 base-pairspecificities have been isolated and these are widely used in geneticmapping, but these enzymes are few in number, are limited to therecognition of G+C-rich sequences, and cleave at sites that tend to behighly clustered (Barlow and Lehrach, Trends Genet., 3:167 [1987]).Recently, endonucleases encoded by group I introns have been discoveredthat might have greater than 12 base-pair specificity (Perlman andButow, Science 246:1106 [1989]), but again, these are few in number.

If the change is not in a restriction enzyme recognition sequence, thenallele-specific oligonucleotides (ASOs), can be designed to hybridize inproximity to the unknown nucleotide, such that a primer extension orligation event can be used as the indicator of a match or a mis-match.Hybridization with radioactively labeled ASOs also has been applied tothe detection of specific point mutations (Conner, Proc. Natl. Acad.Sci., 80:278 [1983]). The method is based on the differences in themelting temperature of short DNA fragments differing by a singlenucleotide (Wallace et al., Nucl. Acids Res., 6:3543 [19791]).Similarly, hybridization with large arrays of short oligonucleotides isnow used as a method for DNA sequencing (Bains and Smith, J. Theor.Biol., 135:303 [19881]) (Drmanac et al., Genomics 4:114 [1989]). Toperform either method it is necessary to work under conditions in whichthe formation of mismatched duplexes is eliminated or reduced whileperfect duplexes still remain stable. Such conditions are termed “highstringency” conditions. The stringency of hybridization conditions canbe altered in a number of ways known in the art. In general, changes inconditions that enhance the formation of nucleic acid duplexes, such asincreases in the concentration of salt, or reduction in the temperatureof the solution, are considered to reduce the stringency of thehybridization conditions. Conversely, reduction of salt and elevation oftemperature are considered to increase the stringency of the conditions.Because it is easy to change and control, variation of the temperatureis commonly used to control the stringency of nucleic acid hybridizationreactions.

Discrimination of hybridization based solely on the presence of amismatch imposes a limit on probe length because effect of a singlemismatch on the stability of a duplex is smaller for longer duplexes.For oligonucleotides designed to detect mutations in genomes of highcomplexity, such as human DNA, it has been shown that the optimal lengthfor hybridization is between 16 and 22 nucleotides, and the temperaturewindow within which the hybridization stringency will allow single basediscrimination can be as large as 10° C. (Wallace [1979], supra).Usually, however, it is much narrower, and for some mismatches, such asG-T, it may be as small as 1 to 2° C. These windows may be even smallerif any other reaction conditions, such as temperature, pH, concentrationof salt and the presence of destabilizing agents (e.g., urea, formamide,dimethylsulfoxide) alter the stringency. Thus, for successful detectionof mutations using such high stringency hybridization methods, a tightcontrol of all parameters affecting duplex stability is critical.

In addition to the degree of homology between the oligonucleotide probeand the target nucleic acid, efficiency of hybridization also depends onthe secondary structure of the target molecule. Indeed, if the region ofthe target molecule that is complementary to the probe is involved inthe formation of intramolecular structures with other regions of thetarget, this will reduce the binding efficiency of the probe.Interference with hybridization by such secondary structure is anotherreason why high stringency conditions are so important for sequenceanalysis by hybridization. High stringency conditions reduce theprobability of secondary structure formation (Gamper et al., J. Mol.Biol., 197:349 [1987]). Another way to of reducing the probability ofsecondary structure formation is to decrease the length of targetmolecules, so that fewer intrastrand interactions can occur. This can bedone by a number of methods, including enzymatic, chemical or thermalcleavage or degradation. Currently, it is standard practice to performsuch a step in commonly used methods of sequence analysis byhybridization to fragment the target nucleic acid into shortoligonucleotides (Fodor et al., Nature 364:555 [1993]).

ASOs have also been adapted to the PCR method. In this, or in any primerextension-based assay, the nucleotide to be investigated is positionedopposite the 3′ end of a primer oligonucleotide. If the bases arecomplementary, then a DNA polymerase can extend the primer with ease; ifthe bases are mismatched, the extension may be blocked. Blocking of PCRby this method has had some degree of success, but not all mismatchesare able to block extension. In fact, a “T” residue on the 3′ end of aprimer can be extended with reasonable efficiency when mis-paired withany of the non-complementary nucleotide when Taq DNA polymerase, acommon PCR enzyme, is used (Kwok, et al., Nucl. Acids. Res. 18:999[1990]). Further, if any of the enzymes having 3′-5′ exonuclease“proofreading” activity (e.g., Vent DNA polymerase, New England Biolabs,Beverly Mass.) are used, the mismatch is first removed, then filled inwith a matched nucleotide before further extension. This dramaticallylimits the scope of application of PCR in this type of direct mutationidentification.

Two other methods of mutation detection rely on detecting changes inelectrophoretic mobility in response to minor sequence changes. One ofthese methods, termed “Denaturing Gradient Gel Electrophoresis” (DGGE)is based on the observation that slightly different sequences willdisplay different patterns of local melting when electrophoreticallyresolved on a gradient gel. In this manner, variants can bedistinguished, as differences in the melting properties of homoduplexesversus heteroduplexes differing in a single nucleotide can be used todetect the presence of mutations in the target sequences because of thecorresponding changes in the electrophoretic mobilities of the hetero-and homoduplexes. The fragments to be analyzed, usually PCR products,are “clamped” at one end by a long stretch of G-C base pairs (30-80) toallow complete denaturation of the sequence of interest without completedissociation of the strands. The attachment of a GC “clamp” to the DNAfragments increases the fraction of mutations that can be recognized byDGGE (Abrams el al., Genomics 7:463 [1990]). Attaching a GC clamp to oneprimer is critical to ensure that the amplified sequence has a lowdissociation temperature (Sheffield et al., Proc. Natl. Acad. Sci.,86:232 [1989]; and Lerman and Silverstein, Meth. Enzymol., 155:482[1987]). Modifications of the technique have been developed, usingtemperature gradient gels (Wartell et al., Nucl. Acids Res.,18:2699-2701 [1990]), and the method can be also applied to RNA:RNAduplexes (Smith et al., Genomics 3:217 [1988]).

Limitations on the utility of DGGE include the requirement that thedenaturing conditions must be optimized for each specific nucleic acidsequence to be tested. Furthermore, the method requires specializedequipment to prepare the gels and maintain the high temperaturesrequired during electrophoresis. The expense associated with thesynthesis of the clamping tail on one oligonucleotide for each sequenceto be tested is also a major consideration. In addition, long runningtimes are required for DGGE. The long running time of DGGE was shortenedin a modification of DGGE called constant denaturant gel electrophoresis(CDGE) (Borrensen et al., Proc. Natl. Acad. Sci. USA 88:8405 [1991]).CDGE requires that gels be performed under different denaturantconditions in order to reach high efficiency for the detection ofunknown mutations. Both DGGE and CDGE are unsuitable for use in clinicallaboratories.

A technique analogous to DGGE, termed temperature gradient gelelectrophoresis (TGGE), uses a thermal gradient rather than a chemicaldenaturant gradient (Scholz et al., Hum. Mol. Genet., 2:2155 [1993]).TGGE requires the use of specialized equipment which can generate atemperature gradient perpendicularly oriented relative to the electricalfield. TGGE can detect mutations in relatively small fragments of DNAtherefore scanning of large gene segments requires the use of multiplePCR products prior to running the gel.

Another common method, called “Single-Strand Conformation Polymorphism”(SSCP) was developed by Hayashi, Sekya and colleagues (reviewed byHayashi, PCR Meth. Appl., 1:34-38, [1991]) and is based on theobservation that single strands of nucleic acid can take oncharacteristic conformations under non-denaturing conditions, and theseconformations influence electrophoretic mobility. The complementarystrands assume sufficiently different structures that the two strandsmay be resolved from one another. Changes in the sequence of a givenfragment will also change the conformation, consequently altering themobility and allowing this to be used as an assay for sequencevariations (Orita, el al., Genomics 5:874 [1989]).

The SSCP process involves denaturing a DNA segment (e.g., a PCR product)that is usually labeled on both strands, followed by slowelectrophoretic separation on a non-denaturing polyacrylamide gel, sothat intra-molecular interactions can form and not be disturbed duringthe run. This technique is extremely sensitive to variations in gelcomposition and temperature. A serious limitation of this method is therelative difficulty encountered in comparing data generated in differentlaboratories, under apparently similar conditions.

The dideoxy fingerprinting (ddF) technique is another techniquedeveloped to scan genes for the presence of unknown mutations (Liu andSommer, PCR Methods Applic, 4:97 [1994]). The ddF technique combinescomponents of Sanger dideoxy sequencing with SSCP. A dideoxy sequencingreaction is performed using one dideoxy terminator and then the reactionproducts are electrophoresed on nondenaturing polyacrylamide gels todetect alterations in mobility of the termination segments as in SSCPanalysis. While ddF is an improvement over SSCP in terms of increasedsensitivity, ddF requires the use of expensive dideoxynucleotides andthis technique is still limited to the analysis of fragments of the sizesuitable for SSCP (i.e., fragments of 200-300 bases for optimaldetection of mutations).

In addition to the above limitations, all of these methods are limitedas to the size of the nucleic acid fragment that can be analyzed. Forthe direct sequencing approach, sequences of greater than 600 base pairsrequire cloning, with the consequent delays and expense of eitherdeletion sub-cloning or primer walking, in order to cover the entirefragment. SSCP and DGGE have even more severe size limitations. Becauseof reduced sensitivity to sequence changes, these methods are notconsidered suitable for larger fragments. Although SSCP is reportedlyable to detect 90% of single-base substitutions within a 200 base-pairfragment, the detection drops to less than 50% for 400 base pairfragments. Similarly, the sensitivity of DGGE decreases as the length ofthe fragment reaches 500 base-pairs. The ddF technique, as a combinationof direct sequencing and SSCP, is also limited by the relatively smallsize of the DNA that can be screened.

Another method of detecting sequence polymorphisms based on theconformation assumed by strands of nucleic acid is the Cleavase®Fragment Length Polymorphism (CFLP®) method (Brow et al., J. Clin.Microbiol., 34:3129 [1996]; PCT International Application No.PCT/US95/14673 [WO 96/15267]; co-pending application Ser. Nos.08/484,956 and 08/520,946). This method uses the actions of a structurespecific nuclease to cleave the folded structures, thus creating a setof product fragments that can by resolved by size (e.g., byelectrophoresis). This method is much less sensitive to size so thatentire genes, rather than gene fragments, may be analyzed.

In many situations (e.g., in many clinical laboratories),electrophoretic separation and analysis may not be technically feasible,or may not be able to accommodate the processing of a large number ofsamples in a cost-effective manner. There is a clear need for a methodof analyzing the characteristic conformations of nucleic acids withoutthe need for either electrophoretic separation of conformations orfragments or for elaborate and expensive methods of visualizing gels(e.g., darkroom supplies, blotting equipment or fluorescence imagers).

In addition to the apparently fortuitous folded conformations that maybe assumed, by any nucleic acid segment, as noted above, the foldedstructures assumed by some nucleic acids are linked in a variety of waysto the function of that nucleic acid. For example, tRNA structure iscritical to its proper function in protein assembly, ribosomal RNA(rRNA) structures are essential to the correct function of the ribosome,and correct folding is essential to the catalytic function of Group Iself-splicing introns (See e.g., the chapters by Woese and Pace (p. 91),Noller (p. 137) and Cech (p. 239) in Gesteland and Atkins (eds.), TheRNA World, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.[1993]). Folded structures in viral RNAs have been linked to infectivity(Proutski et al., J Gen Virol., 78( Pt 7):1543-1549 [1997], alteredsplicing (Ward, et al., Virus Genes 10:91 [1995]), translationalframeshifting (Bidou et al., RNA 3:1153 [1997]), packaging (Miller, etal. J Virol., 71:7648 [1997]), and other functions. In both prokaryotesand eukaryotes, RNA structures are linked to post-transcriptionalcontrol of gene expression through mechanisms including attenuation oftranslation (Girelli et al., Blood 90:2084 [1997], alternative splicing(Howe and Ares, Proc. Natl. Acad. Sci. USA 94:12467 [1997]) andsignaling for RNA degradation (Veyrune et al, Oncogene 11:2127 [1995]).Messenger RNA secondary structure has also been associated withlocalization of that RNA within cells (Serano and Cohen, Develop.,121:3809-3818 [1995]). In DNA it has been shown that cruciformstructures have also been tied to control of gene expression (Hanke etal., J. Mol. Biol., 246:63 [1995]). It can be seen from these fewexamples that the use of folded structures as signals within organismsis not uncommon, nor is it limited to non-protein-encoding RNAs, such asrRNAs, or to non-protein-encoding regions of genomes or messenger RNAs.

Some mutations and polymorphisms associated with altered phenotype actby altering structures assumed by nucleic acids. Any of the functionsand pathways cited above may be altered, e.g., decreased or increased inefficacy, by such a structural alteration. Such alterations in functionmay be associated with medically relevant effects, including but notlimited to tumor growth or morphology (Thompson et al., Oncogene 14:1715[1997]), drug resistance or virulence (Mangada and Igarishi, Virus Genes14:5 [1997], Ward et al., supra) in pathogens. For example, the ironavailability in blood in controlled by the protein ferritin, an ironstorage protein. Ferritin levels are controlled post-transcriptionallyby binding of iron-regulatory proteins to a structure (aniron-responsive element, or IRE) on 5′ untranslated region of theferritin mRNA, thereby blocking translation when iron levels are low.Hereditary hyperferritinemia, an iron storage disorder linked tocataract formation, had been found in some cases to be caused bymutations in the IRE that alter or delete the structure, preventingtranslational regulation.

It can easily be appreciated from these few examples that ability torapidly analyze nucleic acid structure would be a useful tool for bothbasic and clinical research and for diagnostics. Further, accurateidentification of nucleic acid structures would facilitate the designand application of therapeutic agents targeted directly at nucleicacids, such as antisense oligonucleotides, aptamers and peptide nucleicacid agents. The present invention provides methods for designingoligonucleotides that will interact with folded nucleic acids. It iscontemplated that such oligonucleotides may be used for eitherdiagnostic (i.e., detection or analysis of structure) or therapeutic(i.e., alteration of structure function) purposes. When used to detectnucleic acid structure, it is contemplated that the resultingoligonucleotide/folded nucleic acid target complexes may be detecteddirectly (e.g., by capture), or may be detected as the result of afurther catalyzed reaction that is enabled by the complex formation,including but not limited to a ligation, a primer extension, or anuclease cleavage reaction. It will easily be appreciated by thoseskilled in the art that performance of bridging oligonucleotides inthese basic enzymatic reactions is indicative of their utility in assaysthat are based on reiterative performance of these basic reactions,including but not limited to cycle sequencing, polymerase chainreaction, ligase chain reaction, cycling probe reaction and the Invader™invasive cleavage reaction. The present invention provides methods ofusing the bridging oligonucleotides in each of the basic enzymaticreaction systems, and in the Invader™ invasive cleavage system.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for treatingnucleic acid, and in particular, methods and compositions for detectionand characterization of nucleic acid sequences and sequence changes. Thepresent invention provides methods for examining the conformationsassumed by single strands of nucleic acid, forming the basis of novelmethods of detection of specific nucleic acid sequences. The presentinvention contemplates use of novel detection methods for, among otheruses, clinical diagnostic purposes, including but not limited to thedetection and identification of pathogenic organisms.

The present invention contemplates using the interactions between probeoligonucleotides and folded nucleic acid strands in methods fordetection and characterization of nucleic acid sequences and sequencechanges. In another embodiment, the present invention contemplates theuse of structure based nucleic acid interactions in the analysis ofparticular structured regions of nucleic acids, as a determination offunction or alteration of function. A complex formed by the specificinteraction (i.e., reproducible and predictable under a given set ofreaction conditions) of a probe with a target nucleic acid sequence isreferred to herein as a “probe/folded target nucleic acid complex.” Theinteractions contemplated may be a combination of standard hybridizationof oligonucleotides to contiguous, co-linear complementary bases, or mayinclude standard basepairing to non-contiguous regions ofcomplementarity on a strand of nucleic acid to be analyzed. In thiscontext, the term “standard base pairing” refers to hydrogen bondingthat occurs between complementary bases, adenosine to thymidine oruracil and guanine to cytosine to form double helical structures of theA or B form. Such standard base pairing may also be referred to asWatson-Crick base pairing. It is contemplated that the interactionsbetween the oligonucleotides of the present invention (i.e., the probesand the targets) may include non-standard nucleic acid interactionsknown in the art, such as triplex structures, quadraplex aggregates, andthe multibase hydrogen bonding such as is observed within nucleic acidtertiary structures, such as those found in tRNAs. It is contemplatedthat in one embodiment, the interactions between the oligonucleotides ofthe present invention may consist primarily of non-standard nucleic acidinteractions. In one embodiment, the specific probe/folded targetnucleic acid complex uses oligonucleotides that lack uniquecomplementarity to each other (e.g., the shorter nucleic acid probelacks segments that are long enough to be complementary to only a singlesite within the longer nucleic acid or its complement).

The present invention contemplates the use of probes that are designedto interact with non-contiguous regions of complementarity. In oneembodiment, such probes are constructed by incorporating within a singleoligonucleotide segments that are complementary to two or morenon-contiguous regions in the target nucleic acid of interest.

In another embodiment, this mixture is present in an aqueous solution.The invention is not limited by the nature of the aqueous solutionemployed. The aqueous solution may contain mono- and divalent ions,non-ionic detergents, buffers, stabilizers, etc.

The present invention provides a method, comprising: a) providing: i) afolded target having a deoxyribonucleic acid (DNA) sequence comprisingone or more double stranded regions and one or more single strandedregions; and ii) one or more oligonucleotide probes complementary to atleast a portion of said folded target; and b) mixing said folded targetand said one or more probes under conditions such that said probehybridizes to said folded target to form a probe/folded target complex.The degree of complementarity between the probes and the target nucleicacids may be complete or partial (e.g., contain at least one mismatchedbase pair). The method is not limited by the nature of the target DNAemployed to provide the folded target DNA. In one embodiment, the targetDNA comprises single-stranded DNA. In another embodiment, the target DNAcomprises double-stranded DNA. Folded target DNAs may be produced fromeither single-stranded or double-stranded target DNAs by denaturing(e.g., heating) the DNA and then permitting the DNA to form intra-strandsecondary structures. The method is not limited by the manner in whichthe folded target DNA is generated. The target DNA may be denatured by avariety of methods known to the art including heating, exposure toalkali, etc. and then permitted to renature under conditions that favorthe formation of intra-strand duplexes (e.g., cooling, diluting the DNAsolution, neutralizing the pH, etc.).

The method is also not limited by the nature of the oligonucleotideprobes; these probes may comprise DNA, RNA, PNA and combinations thereofas well as comprise modified nucleotides, universal bases, adducts, etc.

In a preferred embodiment, the method further comprises detecting thepresence of said probe/folded target complex. When a detection step isemployed either the probe or the target DNA (or both) may comprise alabel (i.e., a detectable moiety); the invention is not limited by thenature of the label employed or the location of the label (i.e., 5′ end,3′ end, internal to the DNA sequence). A wide variety of suitable labelsare known to the art and include fluorescein, tetrachlorofluorescein,hexachlorofluorescein, Cy3, Cy5, digoxigenin, radioisotopes (e.g., ³²P,³⁵S). In another preferred embodiment, the method further comprisesquantitating the amount of probe/folded target complex formed. Themethod is not limited by the means used for quantitification; when alabeled folded target DNA is employed (e.g., fluorescein or ³²P), theart knows means for quantification (e.g., determination of the amount offluorescence or radioactivity present in the probe/folded targetcomplex).

In a preferred embodiment, the probe in the probe/folded target complexis hybridized to a single stranded region of said folded target. Inanother preferred embodiment, the probe comprises an oligonucleotidehaving a moiety that permits its capture by a solid support. Theinvention is not limited by the nature of the moiety employed to permitcapture. Numerous suitable moieties are known to the art, including butnot limited to, biotin, avidin and streptavidin. Further, it is known inthe art that many small compounds, such as fluorescein and digoxigeninmay serve as haptens for specific capture by appropriate antibodies.Protein conjugates may also be used to allow specific capture byantibodies.

In a preferred embodiment the detection of the presence of saidprobe/folded target complex comprises exposing said probe/folded targetcomplex to a solid support under conditions such that said probe iscaptured by said solid support. As discussed in further detail below,numerous suitable solid supports are known to the art (e.g., beads,particles, dipsticks, wafers, chips, membranes or flat surfaces composedof agarose, nylon, plastics such as polystyrenes, glass or silicon) andmay be employed in the present methods.

In a particularly preferred embodiment, the moiety comprises a biotinmoiety and said solid support comprises a surface having a compoundcapable of binding to said biotin moiety, said compound selected fromthe group consisting of avidin and streptavidin.

In another embodiment, the folded target comprises a deoxyribonucleicacid sequence having a moiety that permits its capture by a solidsupport; as discussed above a number of suitable moieties are known andmay be employed in the present method. In yet another embodiment, thedetection of the presence of said probe/folded target complex comprisesexposing said probe/folded target complex to a solid support underconditions such that said folded target is captured by said solidsupport. In a preferred embodiment, the moiety comprises a biotin moietyand said solid support comprises a surface having a compound capable ofbinding to said biotin moiety, said compound selected from the groupconsisting of avidin and streptavidin.

In a preferred embodiment, the probe is attached to a solid support; theprobe is attached to the solid support in such a manner that the probeis available for hybridization with the folded target nucleic acid, theinvention is not limited by the means employed to attach the probe tothe solid support. The probe may be synthesized in situ on the solidsupport or the probe may be attached (post-synthesis) to the solidsupport via a moiety present on the probe (e.g., using a biotinylatedprobe and solid support comprising avidin or streptavidin). In anotherpreferred embodiment, the folded target nucleic acid is attached to asolid support; this may be accomplished for example using moiety presenton the folded target (e.g., using a biotinylated target nucleic acid andsolid support comprising avidin or streptavidin).

The present invention also provides a method, comprising: a) providing:i) a first folded target having a nucleic acid sequence comprising firstand second portions, said first and second portions each comprising oneor more double stranded regions and one or more single stranded regions;ii) a second folded target having a nucleic acid sequence comprising afirst portion that is identical to said first portion of said firstfolded target and a second portion that differs from said second portionof said first folded target because of a variation in nucleic acidsequence relative to said first folded target, said first and secondportions each comprising one or more double stranded regions and one ormore single stranded regions; iii) first and second oligonucleotideprobes, said first oligonucleotide probe complementary to said firstportion of said first and second folded targets and said secondoligonucleotide probe complementary to said second portion of said firstand second folded targets; and iv) a solid support comprising first,second, third and fourth testing zones, each zone capable of capturingand immobilizing said first and second oligonucleotide probes; b)contacting said first folded target with said first oligonucleotideprobe under conditions such that said first probe binds to said firstfolded target to form a probe/folded target complex in a first mixture;c) contacting said first folded target with said second oligonucleotideprobes under conditions such that said second probe binds to said firstfolded target to form a probe/folded target complex in a second mixture;d) contacting said second folded target with said first oligonucleotideprobe to form a third mixture; e) contacting said second folded targetwith said second oligonucleotide probe to form fourth mixture; and f)adding said first, second, third and fourth mixtures to said first,second, third and fourth testing zones of said solid support,respectively, under conditions such that said probes are captured andimmobilized. The degree of complementarity between the probes and thetarget nucleic acids may be complete or partial (e.g., contain at leastone mismatched base pair).

In a preferred embodiment, the first probe in step d) does notsubstantially hybridize to said second folded target; that is while itis not required that absolutely no formation of a first probe/secondfolded target complex occurs, very little of this complex is formed. Inanother preferred embodiment, the hybridization of said first probe instep d) to said second folded target is reduced relative to thehybridization of said first probe in step c) to said first foldedtarget.

The method is not limited by the nature of the first and second targets.The first and second targets may comprise double- or single-stranded DNAor RNA. The method is also not limited by the nature of theoligonucleotide probes; these probes may comprise DNA, RNA, PNA andcombinations thereof as well as comprise modified nucleotides, universalbases, adducts, etc. In a preferred embodiment, the first and secondoligonucleotide probes comprise DNA.

The present invention further provides a method, comprising: a)providing: i) a first folded target having a nucleic acid sequencecomprising first and second portions, said first and second portionseach comprising one or more double stranded regions and one or moresingle stranded regions; ii) a second folded target having a nucleicacid sequence comprising a first portion that is identical to said firstportion of said first folded target and a second portion that differsfrom said second portion of said first folded target because of avariation in nucleic acid sequence relative to said first folded target,said first and second portions each comprising one or more doublestranded regions and one or more single stranded regions; iii) a solidsupport comprising first and second testing zones, each of said zonescomprising immobilized first and second oligonucleotide probes, saidfirst oligonucleotide probe complementary to said first portion of saidfirst and second folded targets and second oligonucleotide probecomplementary to said second portion of said first and second foldedtargets; and b) contacting said first and second folded targets withsaid solid support under conditions such that said first and secondprobes hybridize to said first folded target to form a probe/foldedtarget complex. The invention is not limited by the nature of the firstand second folded targets. The first and second targets may be derivedfrom double- or single-stranded DNA or RNA. The probes may be completelyor partially complementary to the target nucleic acids. The method isalso not limited by the nature of the oligonucleotide probes; theseprobes may comprise DNA, RNA, PNA and combinations thereof as well ascomprise modified nucleotides, universal bases, adducts, etc. In apreferred embodiment, the first and second oligonucleotide probescomprise DNA. The invention is not limited by the nature of the solidsupport employed as discussed above.

In a preferred embodiment, the contacting of step b) comprises addingsaid first folded target to said first testing zone and adding saidsecond folded target to said second testing zone. In another preferredembodiment, the first and second probes are immobilized in separateportions of said testing zones.

In a preferred embodiment, the first probe in said second testing zonedoes not substantially hybridize to said second folded target; that iswhile it is not required that absolutely no formation of a firstprobe/second folded target complex occurs, very little of this complexis formed. In another preferred embodiment, the first probe in saidsecond testing zone hybridizes to said second folded target with areduced efficiency compared to the hybridization of said first probe infirst testing zone to said first folded target.

In one embodiment, the first and second folded targets comprise DNA. Inanother embodiment, the first and second folded targets comprise RNA.

The present invention also provides a method for treating nucleic acid,comprising: a) providing: i) a nucleic acid target and ii) one or moreoligonucleotide probes; b) treating the nucleic acid target and theprobes under conditions such that the target forms one or more foldedstructures and interacts with one or more probes; and c) analyzing thecomplexes formed between the probes and the target. In a preferredembodiment, the method further comprises providing a solid support forthe capture of the target/probe complexes. Such capture may occur afterthe formation of the structures, or either the probe or the target my bebound to the support before complex formation.

The method is not limited by the nature of the nucleic acid targetemployed. In one embodiment, the nucleic acid of step (a) issubstantially single-stranded. In another embodiment, the nucleic acidis RNA or DNA. It is contemplated that the nucleic acid target comprisea nucleotide analog, including but not limited to the group comprising7-deaza-dATP, 7-deaza-dGTP and dUTP. The nucleic acid target may bedouble stranded. When double-stranded nucleic acid targets are employed,the treating of step (b) comprises: i) rendering the double-strandednucleic acid substantially single-stranded; and ii) exposing thesingle-stranded nucleic acid to conditions such that the single-strandednucleic acid has secondary structure. The invention is not limited bythe method employed to render the double-stranded nucleic acidsubstantially single-stranded; a variety of means known to the art maybe employed. A preferred means for rendering double stranded nucleicacid substantially single-stranded is by the use of increasedtemperature.

In a preferred embodiment, the method further comprises the step ofdetecting said one or more target/probe complexes. The invention is notlimited by the methods used for the detection of the complex(es).

It is contemplated that the methods of the present invention be used forthe detection and identification of microorganisms. It is contemplatedthat the microorganism(s) of the present invention be selected from avariety of microorganisms; it is not intended that the present inventionbe limited to any particular type of microorganism. Rather, it isintended that the present invention will be used with organismsincluding, but not limited to, bacteria, fungi, protozoa, ciliates, andviruses. It is not intended that the microorganisms be limited to aparticular genus, species, strain, or serotype. Indeed, it iscontemplated that the bacteria be selected from the group comprising,but not limited to members of the genera Campylobacter, Escherichia,Mycobacterium, Salmonella, Shigella,and Staphylococcus. In one preferredembodiment, the microorganism(s) comprise strains of multi-drugresistant Mycobacterium tuberculosis. It is also contemplated that thepresent invention be used with viruses, including but not limited tohepatitis C virus, human immunodeficiency virus and simianimmunodeficiency virus.

Another embodiment of the present invention contemplates a method fordetecting and identifying strains of microorganisms, comprising thesteps of extracting nucleic acid from a sample suspected of containingone or more microorganisms; and contacting the extracted nucleic acidwith one or more oligonucleotide probes under conditions such that theextracted nucleic acid forms one or more secondary structures andinteracts with one or more probes. In one embodiment, the method furthercomprises the step of capturing the complexes to a solid support. In yetanother embodiment, the method further comprises the step of detectingthe captured complexes. In one preferred embodiment, the presentinvention further comprises comparing the detected from the extractednucleic acid isolated from the sample with separated complexes derivedfrom one or more reference microorganisms. In such a case the sequenceof the nucleic acids from one or more reference microorganisms may berelated but different (e.g., a wild type control for a mutant sequenceor a known or previously characterized mutant sequence).

In an alternative preferred embodiment, the present invention furthercomprises the step of isolating a polymorphic locus from the extractednucleic acid after the extraction step, so as to generate a nucleic acidtarget, wherein the target is contacted with one or more probeoligonucleotides. In one embodiment, the isolation of a polymorphiclocus is accomplished by polymerase chain reaction amplification. In analternate embodiment, the polymerase chain reaction is conducted in thepresence of a nucleotide analog, including but not limited to the groupcomprising 7-deaza-dATP, 7-deaza-dGTP and dUTP. It is contemplated thatthe polymerase chain reaction amplification will employ oligonucleotideprimers matching or complementary to consensus gene sequences derivedfrom the polymorphic locus. In one embodiment, the polymorphic locuscomprises a ribosomal RNA gene. In a particularly preferred embodiment,the ribosomal RNA gene is a 16S ribosomal RNA gene.

The present invention also contemplates a process for creating a recordreference library of genetic fingerprints characteristic (i.e.,diagnostic) of one or more alleles of the various microorganisms,comprising the steps of providing a nucleic acid target derived frommicrobial gene sequences; comprising the steps of extracting nucleicacid from a sample suspected of containing one or more microorganisms;and contacting the extracted nucleic acid with one or moreoligonucleotide probes under conditions such that the extracted nucleicacid forms one or more secondary structures and interacts with one ormore probes; detecting the captured complexes; and maintaining atestable record reference of the captured complexes.

By the term “genetic fingerprint” it is meant that changes in thesequence of the nucleic acid (e.g., a deletion, insertion or a singlepoint substitution) alter both the sequences detectable by standard basepairing, and alter the structures formed, thus changing the profile ofinteractions between the target and the probe oligonucleotides (e.g.,altering the identity of the probes with which interaction occurs and/oraltering the site/s or strength of the interaction). The measure of theidentity of the probes bound and the strength of the interactionsconstitutes an informative profile that can serve as a “fingerprint” ofthe nucleic acid, reflecting the sequence and allowing rapid detectionand identification of variants.

The methods of the present invention allow for simultaneous analysis ofboth strands (e.g., the sense and antisense strands) and are ideal forhigh-level multiplexing. The products produced are amenable toqualitative, quantitative and positional analysis. The present methodsmay be automated and may be practiced in solution or in the solid phase(e.g., on a solid support). The present methods are powerful in thatthey allow for analysis of longer fragments of nucleic acid than currentmethodologies.

The present invention provides a method, comprising: a) providing: i) afolded target having a deoxyribonucleic acid (DNA) sequence comprisingone or more double stranded regions and one or more single strandedregions; and ii) one or more oligonucleotide probes complementary to atleast a portion of the folded target; and b) mixing the folded targetand the one or more probes under conditions such that the probehybridizes to the folded target to form a probe/folded target complex.The degree of complementarity between the probes and the target nucleicacids may be complete or partial (e.g., contain at least one mismatchedbase pair). The method is not limited by the nature of the target DNAemployed to provide the folded target DNA. In one embodiment, the targetDNA comprises single-stranded DNA. In another embodiment, the target DNAcomprises double-stranded DNA. Folded target DNAs may be produced fromeither single-stranded or double-stranded target DNAs by denaturing(e.g., heating) the DNA and then permitting the DNA to form intra-strandsecondary structures. The method is not limited by the manner in whichthe folded target DNA is generated. The target DNA may be denatured by avariety of methods known to the art including heating, exposure toalkali, etc. and then permitted to renature under conditions that favorthe formation of intra-strand duplexes (e.g., cooling, diluting the DNAsolution, neutralizing the pH, etc.).

The method is also not limited by the nature of the oligonucleotideprobes; these probes may comprise DNA, RNA, PNA and combinations thereofas well as comprise modified nucleotides, universal bases, adducts, etc.

In a preferred embodiment, the method further comprises detecting thepresence of the probe/folded target complex. When a detection step isemployed either the probe or the target DNA (or both) may comprise alabel (i.e., a detectable moiety); the invention is not limited by thenature of the label employed or the location of the label (i.e., 5′ end,3′ end, internal to the DNA sequence). A wide variety of suitable labelsare known to the art and include fluorescein, tetrachlorofluorescein,hexachlorofluorescein, Cy3, Cy5, digoxigenin, radioisotopes (e.g., ³²P,³⁵S). In another preferred embodiment, the method further comprisesquantitating the amount of probe/folded target complex formed. Themethod is not limited by the means used for quantification; when alabeled folded target DNA is employed (e.g., fluorescein or ³²P), theart knows means for quantification (e.g., determination of the amount offluorescence or radioactivity present in the probe/folded targetcomplex).

In a preferred embodiment, the probe in the probe/folded target complexis hybridized to a single stranded region of the folded target. Inanother preferred embodiment, the probe comprises an oligonucleotidehaving a moiety that permits its capture by a solid support. Theinvention is not limited by the nature of the moiety employed to permitcapture. Numerous suitable moieties are known to the art, including butnot limited to, biotin, avidin and streptavidin. Further, it is known inthe art that many small compounds, such as fluorescein and digoxigeninmay serve as haptens for specific capture by appropriate antibodies.Protein conjugates may also be used to allow specific capture byantibodies.

In a preferred embodiment the detection of the presence of theprobe/folded target complex comprises exposing the probe/folded targetcomplex to a solid support under conditions such that the probe iscaptured by the solid support. As discussed in further detail below,numerous suitable solid supports are known to the art (e.g., beads,particles, dipsticks, wafers, chips, membranes or flat surfaces composedof agarose, nylon, plastics such as polystyrenes, glass or silicon) andmay be employed in the present methods.

In a particularly preferred embodiment, the moiety comprises a biotinmoiety and the solid support comprises a surface having a compoundcapable of binding to the biotin moiety, the compound selected from thegroup consisting of avidin and streptavidin.

In another embodiment, the folded target comprises a deoxyribonucleicacid sequence having a moiety that permits its capture by a solidsupport; as discussed above a number of suitable moieties are known andmay be employed in the present method. In yet another embodiment, thedetection of the presence of the probe/folded target complex comprisesexposing the probe/folded target complex to a solid support underconditions such that the folded target is captured by the solid support.In a preferred embodiment, the moiety comprises a biotin moiety and thesolid support comprises a surface having a compound capable of bindingto the biotin moiety, the compound selected from the group consisting ofavidin and streptavidin.

In a preferred embodiment, the probe is attached to a solid support; theprobe is attached to the solid support in such a manner that the probeis available for hybridization with the folded target nucleic acid, theinvention is not limited by the means employed to attach the probe tothe solid support. The probe may be synthesized in situ on the solidsupport or the probe may be attached (post-synthesis) to the solidsupport via a moiety present on the probe (e.g., using a biotinylatedprobe and solid support comprising avidin or streptavidin). In anotherpreferred embodiment, the folded target nucleic acid is attached to asolid support; this may be accomplished for example using moiety presenton the folded target (e.g., using a biotinylated target nucleic acid andsolid support comprising avidin or streptavidin).

The present invention also provides a method, comprising: a) providing:i) a first folded target having a nucleic acid sequence comprising firstand second portions, said first and second portions each comprising oneor more double stranded regions, and one or more single strandedregions, and further comprising two or more non-contiguous portions, andone or more intervening regions; ii) a second folded target having anucleic acid sequence comprising a first portion that is identical tosaid first portion of said first folded target and a second portion thatdiffers from said second portion of said first folded target because ofa variation in nucleic acid sequence relative to said first foldedtarget, said first and second portions each comprising one or moredouble stranded regions, and one or more single stranded regions, andfurther comprising two or more non-contiguous portions, and one or moreintervening regions; iii) first and second bridging oligonucleotides,said first bridging oligonucleotide complementary to said two or morenon-contiguous portions of said first portion of said first and secondfolded targets and said second bridging oligonucleotide complementary tosaid two or more non-contiguous portions of said second portion of saidfirst and second folded targets; and iv) a solid support comprisingfirst, second, third and fourth testing zones, each zone capable ofcapturing and immobilizing said first and second bridgingoligonucleotides; b) contacting the first folded target with the firstoligonucleotide probe under conditions such that the first probe bindsto the first folded target to form a probe/folded target complex in afirst mixture; c) contacting the first folded target with the secondoligonucleotide probes under conditions such that the second probe bindsto the first folded target to form a probe/folded target complex in asecond mixture; d) contacting the second folded target with the firstoligonucleotide probe to form a third mixture; e) contacting the secondfolded target with the second oligonucleotide probe to form fourthmixture; and f) adding the first, second, third and fourth mixtures tothe first, second, third and fourth testing zones of the solid support,respectively, under conditions such that the probes are captured andimmobilized. The degree of complementarity between the probes and thetarget nucleic acids may be complete or partial (e.g., contain at leastone mismatched base pair).

In a preferred embodiment, the first probe in step d) does notsubstantially hybridize to the second folded target; that is while it isnot required that absolutely no formation of a first probe/second foldedtarget complex occurs, very little of this complex is formed. In anotherpreferred embodiment, the hybridization of the first probe in step d) tothe second folded target is reduced relative to the hybridization of thefirst probe in step c) to the first folded target.

The method is not limited by the nature of the first and second targets.The first and second targets may comprise double- or single-stranded DNAor RNA. The method is also not limited by the nature of theoligonucleotide probes; these probes may comprise DNA, RNA, PNA andcombinations thereof as well as comprise modified nucleotides, universalbases, adducts, etc. In a preferred embodiment, the first and secondoligonucleotide probes comprise DNA.

The present invention further provides a method, comprising: a)providing: i) a first folded target having a nucleic acid sequencecomprising first and second portions, the first and second portions eachcomprising one or more double stranded regions and one or more singlestranded regions; ii) a second folded target having a nucleic acidsequence comprising a first portion that is identical to the firstportion of the first folded target and a second portion that differsfrom the second portion of the first folded target because of avariation in nucleic acid sequence relative to the first folded target,the first and second portions each comprising one or more doublestranded regions and one or more single stranded regions; iii) a solidsupport comprising first and second testing zones, each of the zonescomprising immobilized first and second oligonucleotide probes, thefirst oligonucleotide probe complementary to the first portion of thefirst and second folded targets and second oligonucleotide probecomplementary to the second portion of the first and second foldedtargets; and b) contacting the first and second folded targets with thesolid support under conditions such that the first and second probeshybridize to the first folded target to form a probe/folded targetcomplex. The invention is not limited by the nature of the first andsecond folded targets. The first and second targets may be derived fromdouble- or single-stranded DNA or RNA. The probes may be completely orpartially complementary to the target nucleic acids. The method is alsonot limited by the nature of the oligonucleotide probes; these probesmay comprise DNA, RNA, PNA and combinations thereof as well as comprisemodified nucleotides, universal bases, adducts, etc. In a preferredembodiment, the first and second oligonucleotide probes comprise DNA.The invention is not limited by the nature of the solid support employedas discussed above.

In a preferred embodiment, the contacting of step b) comprises addingthe first folded target to the first testing zone and adding the secondfolded target to the second testing zone. In another preferredembodiment, the first and second probes are immobilized in separateportions of the testing zones.

In a preferred embodiment, the first probe in the second testing zonedoes not substantially hybridize to the second folded target; that iswhile it is not required that absolutely no formation of a firstprobe/second folded target complex occurs, very little of this complexis formed. In another preferred embodiment, the first probe in thesecond testing zone hybridizes to the second folded target with areduced efficiency compared to the hybridization of the first probe infirst testing zone to the first folded target.

In one embodiment, the first and second folded targets comprise DNA. Inanother embodiment, the first and second folded targets comprise RNA.

The present invention also provides a method for treating nucleic acid,comprising: a) providing: i) a nucleic acid target and ii) one or moreoligonucleotide probes; b) treating the nucleic acid target and theprobes under conditions such that the target forms one or more foldedstructures and interacts with one or more probes; and c) analyzing thecomplexes formed between the probes and the target. In a preferredembodiment, the method further comprises providing a solid support forthe capture of the target/probe complexes. Such capture may occur afterthe formation of the structures, or either the probe or the target my bebound to the support before complex formation.

The method is not limited by the nature of the nucleic acid targetemployed. In one embodiment, the nucleic acid of step (a) issubstantially single-stranded. In another embodiment, the nucleic acidis RNA or DNA. It is contemplated that the nucleic acid target comprisea nucleotide analog, including but not limited to the group comprising7-deaza-dATP, 7-deaza-dGTP and dUTP. The nucleic acid target may bedouble stranded. When double-stranded nucleic acid targets are employed,the treating of step (b) comprises: i) rendering the double-strandednucleic acid substantially single-stranded; and ii) exposing thesingle-stranded nucleic acid to conditions such that the single-strandednucleic acid has secondary structure. The invention is not limited bythe method employed to render the double-stranded nucleic acidsubstantially single-stranded; a variety of means known to the art maybe employed. A preferred means for rendering double stranded nucleicacid substantially single-stranded is by the use of increasedtemperature.

In a preferred embodiment, the method further comprises the step ofdetecting the one or more target/probe complexes. The invention is notlimited by the methods used for the detection of the complex(es).

It is contemplated that the methods of the present invention be used forthe detection and identification of microorganisms. It is contemplatedthat the microorganism(s) of the present invention be selected from avariety of microorganisms; it is not intended that the present inventionbe limited to any particular type of microorganism. Rather, it isintended that the present invention will be used with organismsincluding, but not limited to, bacteria, fungi, protozoa, ciliates, andviruses. It is not intended that the microorganisms be limited to aparticular genus, species, strain, or serotype. Indeed, it iscontemplated that the bacteria be selected from the group comprising,but not limited to members of the genera Campylobacter, Escherichia,Mycobacterium, Salmonella, Shigella, and Staphylococcus. In onepreferred embodiment, the microorganism(s) comprise strains ofmulti-drug resistant Mycobacterium tuberlosis. It is also contemplatedthat the present invention be used with viruses, including but notlimited to hepatitis C virus, human immunodeficiency virus and simianimmunodeficiency virus.

Another embodiment of the present invention contemplates a method fordetecting and identifying strains of microorganisms, comprising thesteps of extracting nucleic acid from a sample suspected of containingone or more microorganisms; and contacting the extracted nucleic acidwith one or more oligonucleotide probes under conditions such that theextracted nucleic acid forms one or more secondary structures andinteracts with one or more probes. In one embodiment, the method furthercomprises the step of capturing the complexes to a solid support. In yetanother embodiment, the method further comprises the step of detectingthe captured complexes. In one preferred embodiment, the presentinvention further comprises comparing the detected from the extractednucleic acid isolated from the sample with separated complexes derivedfrom one or more reference microorganisms. In such a case the sequenceof the nucleic acids from one or more reference microorganisms may berelated but different (e.g., a wild type control for a mutant sequenceor a known or previously characterized mutant sequence).

In an alternative preferred embodiment, the present invention furthercomprises the step of isolating a polymorphic locus from the extractednucleic acid after the extraction step, so as to generate a nucleic acidtarget, wherein the target is contacted with one or more probeoligonucleotides. In one embodiment, the isolation of a polymorphiclocus is accomplished by polymerase chain reaction amplification. In analternate embodiment, the polymerase chain reaction is conducted in thepresence of a nucleotide analog, including but not limited to the groupcomprising 7-deaza-dATP, 7-deaza-dGTP and dUTP. It is contemplated thatthe polymerase chain reaction amplification will employ oligonucleotideprimers matching or complementary to consensus gene sequences derivedfrom the polymorphic locus. In one embodiment, the polymorphic locuscomprises a ribosomal RNA gene. In a particularly preferred embodiment,the ribosomal RNA gene is a 16S ribosomal RNA gene.

The present invention also contemplates a process for creating a recordreference library of genetic fingerprints characteristic (i.e.,diagnostic) of one or more alleles of the various microorganisms,comprising the steps of providing a nucleic acid target derived frommicrobial gene sequences; comprising the steps of extracting nucleicacid from a sample suspected of containing one or more microorganisms;and contacting the extracted nucleic acid with one or moreoligonucleotide probes under conditions such that the extracted nucleicacid forms one or more secondary structures and interacts with one ormore probes; detecting the captured complexes; and maintaining atestable record reference of the captured complexes.

By the term “genetic fingerprint” it is meant that changes in thesequence of the nucleic acid (e.g., a deletion, insertion or a singlepoint substitution) alter both the sequences detectable by standard basepairing, and alter the structures formed, thus changing the profile ofinteractions between the target and the probe oligonucleotides (e.g.,altering the identity of the probes with which interaction occurs and/oraltering the site/s or strength of the interaction). The measure of theidentity of the probes bound and the strength of the interactionsconstitutes an informative profile that can serve as a “fingerprint” ofthe nucleic acid, reflecting the sequence and allowing rapid detectionand identification of variants.

The methods of the present invention allow for simultaneous analysis ofboth strands (e.g., the sense and antisense strands) and are ideal forhigh-level multiplexing. The products produced are amenable toqualitative, quantitative and positional analysis. The present methodsmay be automated and may be practiced in solution or in the solid phase(e.g., on a solid support). The present methods are powerful in thatthey allow for analysis of longer fragments of nucleic acid than currentmethodologies.

The present invention further provides methods for determination ofstructure formation in nucleic acid targets, comprising the steps of: a)providing: i) a folded target having a deoxyribonucleic acid sequencecomprising one or more double stranded regions, and one or more singlestranded regions, and further comprising two or more non-contiguousportions, and one or more intervening regions; and ii) one or morebridging oligonucleotide probes complementary to two or morenon-contiguous portions of the folded target; and b) mixing the foldedtarget and one or more bridging oligonucleotide probes under conditionssuch that the bridging oligonucleotide probes hybridize to the foldedtarget to form a probe/folded target complex.

In preferred embodiments, the one or more intervening regions of thefolded targets comprise at least five nucleotides. In yet otherembodiments, either of the targets and/or either of the bridgingoligonucleotides contain intervening regions comprised of non-nucleotidespacers of any length. In a preferred embodiment, the first and secondoligonucleotide probes comprise DNA. In alternative embodiments, themethod further comprises detecting the presence of the probe/foldedtarget complex. In yet other embodiments, the method further comprisesquantitating the amount of probe/folded target complex formed. In yetother embodiments of the method, the bridging oligonucleotide probe inthe probe/folded target complex is hybridized to at least one singlestranded region of the folded target.

The method is not limited by the nature of the target DNA employed toprovide the folded target DNA, nor is the method limited by the mannerin which the folded target DNA is generated. The method is also notlimited by the nature of the bridging oligonucleotide probes; theseprobes may comprise DNA, RNA, PNA and combinations thereof as well ascomprise modified nucleotides, universal bases, adducts, etc.

In a preferred embodiment, the method further comprises detecting thepresence of the probe/folded target complex. When a detection step isemployed either the bridging oligonucleotide probe or the target DNA (orboth) may comprise a label (i.e., a detectable moiety); the invention isnot limited by the nature of the label employed or the location of thelabel (i.e., 5′ end, 3′ end, internal to the DNA sequence). A widevariety of suitable labels are known to the art and include fluorescein,tetrachlorofluorescein, hexachlorofluorescein, Cy3, Cy5, digoxigenin,radioisotopes (e.g., ³²P, ³⁵S). In another preferred embodiment, themethod further comprises quantitating the amount of probe/folded targetcomplex formed. The method is not limited by the means used forquantification; when a labeled folded target DNA is employed (e.g.,fluorescein or ³²P), the art knows means for quantification (e.g.,determination of the amount of fluorescence or radioactivity present inthe probe/folded target complex).

In another preferred embodiment, the bridging oligonucleotide probecomprises a bridging oligonucleotide having a moiety that permits itscapture by a solid support. The invention is not limited by the natureof the moiety employed to permit capture. Numerous suitable moieties areknown to the art, including but not limited to, biotin, avidin andstreptavidin. Further, it is known in the art that many small compounds,such as fluorescein and digoxigenin may serve as haptens for specificcapture by appropriate antibodies. Protein conjugates may also be usedto allow specific capture by antibodies.

In a preferred embodiment the detection of the presence of theprobe/folded target complex comprises exposing the probe/folded targetcomplex to a solid support under conditions such that the bridgingoligonucleotide probe is captured by the solid support. As discussed infurther detail below, numerous suitable solid supports are known to theart (e.g., beads, particles, dipsticks, wafers, chips, membranes or flatsurfaces composed of agarose, nylon, plastics such as polystyrenes,glass or silicon) and may be employed in the present methods.

In a particularly preferred embodiment, the moiety comprises a biotinmoiety and the solid support comprises a surface having a compoundcapable of binding to the biotin moiety, the compound selected from thegroup consisting of avidin and streptavidin.

In another embodiment, the folded target comprises a deoxyribonucleicacid sequence having a moiety that permits its capture by a solidsupport; as discussed above a number of suitable moieties are known andmay be employed in the present method. In yet another embodiment, thedetection of the presence of the probe/folded target complex comprisesexposing the probe/folded target complex to a solid support underconditions such that the folded target is captured by the solid support.In a preferred embodiment, the moiety comprises a biotin moiety and thesolid support comprises a surface having a compound capable of bindingto the biotin moiety, the compound selected from the group consisting ofavidin and streptavidin.

In a preferred embodiment, the bridging oligonucleotide probe isattached to a solid support; the probe is attached to the solid supportin such a manner that the bridging oligonucleotide probe is availablefor hybridization with the folded target nucleic acid. The invention isnot limited by the means employed to attach the bridging oligonucleotideprobe to the solid support. The bridging oligonucleotide probe may besynthesized in situ on the solid support or the probe may be attached(post-synthesis) to the solid support via a moiety present on thebridging oligonucleotide probe (e.g., using a biotinylated probe andsolid support comprising avidin or streptavidin). In another preferredembodiment, the folded target nucleic acid is attached to a solidsupport; this may be accomplished for example using moiety present onthe folded target (e.g., using a biotinylated target nucleic acid andsolid support comprising avidin or streptavidin).

The present invention also provides methods for analyzing the structureof nucleic acid targets, comprising: a) providing: i) a first foldedtarget having a nucleic acid sequence comprising first and secondportions, the first and second portions each comprising one or moredouble stranded regions and one or more single stranded regions; ii) asecond folded target having a nucleic acid sequence comprising a firstportion that is identical to the first portion of the first foldedtarget and a second portion that differs from the second portion of thefirst folded target because of a variation in nucleic acid sequencerelative to the first folded target, the first and second portions eachcomprising one or more double stranded regions and one or more singlestranded regions; iii) first and second bridging oligonucleotides,wherein the first bridging oligonucleotide is complementary to the firstportion of the first and second folded targets and the second bridgingoligonucleotide is complementary to the second portion of the first andsecond folded targets; and iv) a solid support comprising first, second,third and fourth testing zones, each zone capable of capturing andimmobilizing the first and second bridging oligonucleotides; b)contacting the first folded target with the first bridgingoligonucleotide under conditions such that the first bridgingoligonucleotide binds to the first folded target to form a probe/foldedtarget complex in a first mixture; c) contacting the first folded targetwith the second bridging oligonucleotide under conditions such that thesecond bridging oligonucleotide binds to the first folded target to forma probe/folded target complex in a second mixture; d) contacting thesecond folded target with the first bridging oligonucleotide to form athird mixture; e) contacting the second folded target with the secondbridging oligonucleotide to form fourth mixture; and f) adding thefirst, second, third and fourth mixtures to the first, second, third andfourth testing zones of the solid support, respectively, underconditions such that the first and second bridging oligonucleotides arecaptured and immobilized.

The method is not limited by the nature of the first and second targets.The first and/or second target may comprise one or more non-contiguousregions, as well as one or more intervening regions. In preferredembodiments, the intervening regions comprise at least five nucleotides.The method is also not limited by the nature of the bridgingoligonucleotide probes; these bridging oligonucleotide probes maycomprise DNA, RNA, PNA and combinations thereof as well as comprisemodified nucleotides, universal bases, adducts, etc. In someembodiments, the first and/or second bridging oligonucleotide probescomprise one or more intervening regions. In alternative embodiments,the intervening region of the bridging oligonucleotide probes comprisesat least two nucleotides. In yet other embodiments, either of thetargets and/or either of the bridging oligonucleotides containintervening regions comprised of non-nucleotide spacers of any length.In a preferred embodiment, the first and second oligonucleotide probescomprise DNA. In a preferred embodiment, the first and second bridgingoligonucleotide probes comprise DNA.

In alternative embodiments, the first bridging oligonucleotide in stepd) does not substantially hybridize to the second folded target. In yetanother embodiment, the hybridization of the first bridgingoligonucleotide in step d) to the second folded target is reducedrelative to the hybridization of the first bridging oligonucleotide instep c) to the first folded target. In further embodiments, the firstand second targets comprise DNA, and/or the first and second bridgingoligonucleotides comprise DNA.

The present invention also provides methods for analyzing folded nucleicacid targets, comprising: a) providing: i) a first folded target havinga nucleic acid sequence comprising first and second portions, whereinthe first and second portions each comprise one or more double strandedregions and one or more single stranded regions; ii) a second foldedtarget having a nucleic acid sequence comprising a first portion that isidentical to the first portion of the first folded target, and a secondportion that differs from the second portion of the first folded targetbecause of a variation in nucleic acid sequence relative to the firstfolded target, the first and second portions each comprising one or moredouble stranded regions and one or more single stranded regions; iii) asolid support comprising first and second testing zones, each of thezones comprising immobilized first and second bridging oligonucleotides,the first bridging oligonucleotide being complementary to the firstportion of the first and second folded targets and second bridgingoligonucleotide being complementary to the second portion of the firstand second folded targets; and b) contacting the first and second foldedtargets with the solid support under conditions such that the first andsecond bridging oligonucleotides hybridize to the first folded target toform a probe/folded target complex.

In some embodiments, the contacting of step b) comprises adding thefirst folded target to the first testing zone and adding the secondfolded target to the second testing zone. In alternative embodiments,the first and second bridging oligonucleotides are immobilized inseparate portions of the testing zones. In yet other embodiments, thefirst bridging oligonucleotide in the second testing zone does notsubstantially hybridize to the second folded target. In furtherembodiments, the first bridging oligonucleotide in the second testingzone hybridizes to the second folded target with a reduced efficiencycompared to the hybridization of the first bridging oligonucleotide infirst testing zone to the first folded target.

The method is not limited by the nature of, nor the method of generatingthe first and second folded targets. The method is also not limited bythe nature of, or the method of generating the oligonucleotide probes;these probes may comprise DNA, RNA, PNA and combinations thereof as wellas comprise modified nucleotides, universal bases, adducts, etc. In someembodiments, the first and/or second folded target comprises one or moreintervening region comprised of at least five nucleotides. In yet otherembodiments, the first and/or second bridging oligonucleotide probecomprises one or more intervening regions comprised of at least twonucleotides. In yet other embodiments, either of the targets and/oreither of the bridging oligonucleotides contain intervening regionscomprised of non-nucleotide spacers of any length. In a preferredembodiment, the first and second oligonucleotide probes comprise DNA.The invention is not limited by the nature of the solid support employedas discussed above. In some preferred embodiments of the method, thefirst and second folded targets comprise DNA. In alternativeembodiments, the first and second folded targets comprise RNA. In yetother embodiments, the first and second bridging oligonucleotidescomprise DNA.

In one embodiment, the present invention provides a method, comprising:a) providing: i) a folded target having a deoxyribonucleic acid (DNA)sequence comprising one or more double stranded regions and one or moresingle stranded regions; and ii) one or more oligonucleotide probescomplementary to at least a portion of the folded target; and b) mixingthe folded target and the one or more probes under conditions such thatthe probe hybridizes to the folded target to form a probe/folded targetcomplex. The degree of complementarity between the probes and the targetnucleic acids may be complete or partial (e.g., contain at least onemismatched base pair). The method is not limited by the nature of thetarget DNA employed to provide the folded target DNA. In one embodiment,the target DNA comprises single-stranded DNA. In another embodiment, thetarget DNA comprises double-stranded DNA. Folded target DNAs may beproduced from either single-stranded or double-stranded target DNAs bydenaturing (e.g., heating) the DNA and then permitting the DNA to formintra-strand secondary structures. The method is not limited by themanner in which the folded target DNA is generated. The target DNA maybe denatured by a variety of methods known to the art including heating,exposure to alkali, etc. and then permitted to renature under conditionsthat favor the formation of intra-strand duplexes (e.g., cooling,diluting the DNA solution, neutralizing the pH, etc.).

The method is also not limited by the nature of the oligonucleotideprobes; these probes may comprise DNA, RNA, PNA and combinations thereofas well as comprise modified nucleotides, universal bases, adducts, etc.

In a preferred embodiment, the method further comprises detecting thepresence of the probe/folded target complex. When a detection step isemployed either the probe or the target DNA (or both) may comprise alabel (i.e., a detectable moiety); the invention is not limited by thenature of the label employed or the location of the label (i.e., 5′ end,3′ end, internal to the DNA sequence). A wide variety of suitable labelsare known to the art and include fluorescein, tetrachlorofluorescein,hexachlorofluorescein, Cy3, Cy5, digoxigenin, radioisotopes (e.g., ³²P,³⁵S). In another preferred embodiment, the method further comprisesquantitating the amount of probe/folded target complex formed. Themethod is not limited by the means used for quantification; when alabeled folded target DNA is employed (e.g., fluorescein or 32p), theart knows means for quantification (e.g., determination of the amount offluorescence or radioactivity present in the probe/folded targetcomplex).

In a preferred embodiment, the probe in the probe/folded target complexis hybridized to a single stranded region of the folded target. Inanother preferred embodiment, the probe comprises an oligonucleotidehaving a moiety that permits its capture by a solid support. Theinvention is not limited by the nature of the moiety employed to permitcapture. Numerous suitable moieties are known to the art, including butnot limited to, biotin, avidin and streptavidin. Further, it is known inthe art that many small compounds, such as fluorescein and digoxigeninmay serve as haptens for specific capture by appropriate antibodies.Protein conjugates may also be used to allow specific capture byantibodies.

In a preferred embodiment the detection of the presence of theprobe/folded target complex comprises exposing the probe/folded targetcomplex to a solid support under conditions such that the probe iscaptured by the solid support. As discussed in further detail below,numerous suitable solid supports are known to the art (e.g., beads,particles, dipsticks, wafers, chips, membranes or flat surfaces composedof agarose, nylon, plastics such as polystyrenes, glass or silicon) andmay be employed in the present methods.

In a particularly preferred embodiment, the moiety comprises a biotinmoiety and the solid support comprises a surface having a compoundcapable of binding to the biotin moiety, the compound selected from thegroup consisting of avidin and streptavidin.

In another embodiment, the folded target comprises a deoxyribonucleicacid sequence having a moiety that permits its capture by a solidsupport; as discussed above a number of suitable moieties are known andmay be employed in the present method. In yet another embodiment, thedetection of the presence of the probe/folded target complex comprisesexposing the probe/folded target complex to a solid support underconditions such that the folded target is captured by the solid support.In a preferred embodiment, the moiety comprises a biotin moiety and thesolid support comprises a surface having a compound capable of bindingto the biotin moiety, the compound selected from the group consisting ofavidin and streptavidin.

In a preferred embodiment, the probe is attached to a solid support; theprobe is attached to the solid support in such a manner that the probeis available for hybridization with the folded target nucleic acid, theinvention is not limited by the means employed to attach the probe tothe solid support. The probe may be synthesized in situ on the solidsupport or the probe may be attached (post-synthesis) to the solidsupport via a moiety present on the probe (e.g., using a biotinylatedprobe and solid support comprising avidin or streptavidin). In anotherpreferred embodiment, the folded target nucleic acid is attached to asolid support; this may be accomplished for example using moiety presenton the folded target (e.g., using a biotinylated target nucleic acid andsolid support comprising avidin or streptavidin).

The present invention also provides a method, comprising: a) providing:i) a first folded target having a nucleic acid sequence comprising firstand second portions, the first and second portions each comprising oneor more double stranded regions and one or more single stranded regions;ii) a second folded target having a nucleic acid sequence comprising afirst portion that is identical to the first portion of the first foldedtarget and a second portion that differs from the second portion of thefirst folded target because of a variation in nucleic acid sequencerelative to the first folded target, the first and second portions eachcomprising one or more double stranded regions and one or more singlestranded regions; iii) first and second oligonucleotide probes, thefirst oligonucleotide probe complementary to the first portion of thefirst and second folded targets and the second oligonucleotide probecomplementary to the second portion of the first and second foldedtargets; and iv) a solid support comprising first, second, third andfourth testing zones, each zone capable of capturing and immobilizingthe first and second oligonucleotide probes; b) contacting the firstfolded target with the first oligonucleotide probe under conditions suchthat the first probe binds to the first folded target to form aprobe/folded target complex in a first mixture; c) contacting the firstfolded target with the second oligonucleotide probes under conditionssuch that the second probe binds to the first folded target to form aprobe/folded target complex in a second mixture; d) contacting thesecond folded target with the first oligonucleotide probe to form athird mixture; e) contacting the second folded target with the secondoligonucleotide probe to form fourth mixture; and f) adding the first,second, third and fourth mixtures to the first, second, third and fourthtesting zones of the solid support, respectively, under conditions suchthat the probes are captured and immobilized. The degree ofcomplementarity between the probes and the target nucleic acids may becomplete or partial (e.g., contain at least one mismatched base pair).

In a preferred embodiment, the first probe in step d) does notsubstantially hybridize to the second folded target; that is while it isnot required that absolutely no formation of a first probe/second foldedtarget complex occurs, very little of this complex is formed. In anotherpreferred embodiment, the hybridization of the first probe in step d) tothe second folded target is reduced relative to the hybridization of thefirst probe in step c) to the first folded target.

The method is not limited by the nature of the first and second targets.The first and second targets may comprise double- or single-stranded DNAor RNA. The method is also not limited by the nature of theoligonucleotide probes; these probes may comprise DNA, RNA, PNA andcombinations thereof as well as comprise modified nucleotides, universalbases, adducts, etc. In a preferred embodiment, the first and secondoligonucleotide probes comprise DNA.

The present invention further provides a method, comprising: a)providing: i) a first folded target having a nucleic acid sequencecomprising first and second portions, the first and second portions eachcomprising one or more double stranded regions and one or more singlestranded regions; ii) a second folded target having a nucleic acidsequence comprising a first portion that is identical to the firstportion of the first folded target and a second portion that differsfrom the second portion of the first folded target because of avariation in nucleic acid sequence relative to the first folded target,the first and second portions each comprising one or more doublestranded regions and one or more single stranded regions; iii) a solidsupport comprising first and second testing zones, each of the zonescomprising immobilized first and second oligonucleotide probes, thefirst oligonucleotide probe complementary to the first portion of thefirst and second folded targets and second oligonucleotide probecomplementary to the second portion of the first and second foldedtargets; and b) contacting the first and second folded targets with thesolid support under conditions such that the first and second probeshybridize to the first folded target to form a probe/folded targetcomplex. The invention is not limited by the nature of the first andsecond folded targets. The first and second targets may be derived fromdouble- or single-stranded DNA or RNA. The probes may be completely orpartially complementary to the target nucleic acids. The method is alsonot limited by the nature of the oligonucleotide probes; these probesmay comprise DNA, RNA, PNA and combinations thereof as well as comprisemodified nucleotides, universal bases, adducts, etc. In a preferredembodiment, the first and second oligonucleotide probes comprise DNA.The invention is not limited by the nature of the solid support employedas discussed above.

In a preferred embodiment, the contacting of step b) comprises addingthe first folded target to the first testing zone and adding the secondfolded target to the second testing zone. In another preferredembodiment, the first and second probes are immobilized in separateportions of the testing zones.

In a preferred embodiment, the first probe in the second testing zonedoes not substantially hybridize to the second folded target; that iswhile it is not required that absolutely no formation of a firstprobe/second folded target complex occurs, very little of this complexis formed. In another preferred embodiment, the first probe in thesecond testing zone hybridizes to the second folded target with areduced efficiency compared to the hybridization of the first probe infirst testing zone to the first folded target.

In one embodiment, the first and second folded targets comprise DNA. Inanother embodiment, the first and second folded targets comprise RNA.

The present invention also provides a method for treating nucleic acid,comprising: a) providing: i) a nucleic acid target and ii) one or moreoligonucleotide probes; b) treating the nucleic acid target and theprobes under conditions such that the target forms one or more foldedstructures and interacts with one or more probes; and c) analyzing thecomplexes formed between the probes and the target. In a preferredembodiment, the method further comprises providing a solid support forthe capture of the target/probe complexes. Such capture may occur afterthe formation of the structures, or either the probe or the target my bebound to the support before complex formation.

The method is not limited by the nature of the nucleic acid targetemployed. In one embodiment, the nucleic acid of step (a) issubstantially single-stranded. In another embodiment, the nucleic acidis RNA or DNA. It is contemplated that the nucleic acid target comprisea nucleotide analog, including but not limited to the group comprising7-deaza-dATP, 7-deaza-dGTP and dUTP. The nucleic acid target may bedouble stranded. When double-stranded nucleic acid targets are employed,the treating of step (b) comprises: i) rendering the double-strandednucleic acid substantially single-stranded; and ii) exposing thesingle-stranded nucleic acid to conditions such that the single-strandednucleic acid has secondary structure. The invention is not limited bythe method employed to render the double-stranded nucleic acidsubstantially single-stranded; a variety of means known to the art maybe employed. A preferred means for rendering double stranded nucleicacid substantially single-stranded is by the use of increasedtemperature.

In a preferred embodiment, the method further comprises the step ofdetecting the one or more target/probe complexes. The invention is notlimited by the methods used for the detection of the complex(es).

It is contemplated that the methods of the present invention be used forthe detection and identification of microorganisms. It is contemplatedthat the microorganism(s) of the present invention be selected from avariety of microorganisms; it is not intended that the present inventionbe limited to any particular type of microorganism. Rather, it isintended that the present invention will be used with organismsincluding, but not limited to, bacteria, fungi, protozoa, ciliates, andviruses. It is not intended that the microorganisms be limited to aparticular genus, species, strain, or serotype. Indeed, it iscontemplated that the bacteria be selected from the group comprising,but not limited to members of the genera Campylobacter, Escherichia,Mycobacterium, Salmonella, Shigella, and Staphylococcus. In onepreferred embodiment, the microorganism(s) comprise strains ofmulti-drug resistant Mycobacterium tuberlosis. It is also contemplatedthat the present invention be used with viruses, including but notlimited to hepatitis C virus, human immunodeficiency virus, simianimmunodeficiency virus, and influenza virus (e.g., influenza type A)

Another embodiment of the present invention contemplates a method fordetecting and identifying strains of microorganisms, comprising thesteps of extracting nucleic acid from a sample suspected of containingone or more microorganisms; and contacting the extracted nucleic acidwith one or more oligonucleotide probes under conditions such that theextracted nucleic acid forms one or more secondary structures andinteracts with one or more probes. In one embodiment, the method furthercomprises the step of capturing the complexes to a solid support. In yetanother embodiment, the method further comprises the step of detectingthe captured complexes. In one preferred embodiment, the presentinvention further comprises comparing the detected from the extractednucleic acid isolated from the sample with separated complexes derivedfrom one or more reference microorganisms. In such a case the sequenceof the nucleic acids from one or more reference microorganisms may berelated but different (e.g., a wild type control for a mutant sequenceor a known or previously characterized mutant sequence).

In an alternative preferred embodiment, the present invention furthercomprises the step of isolating a polymorphic locus from the extractednucleic acid after the extraction step, so as to generate a nucleic acidtarget, wherein the target is contacted with one or more probeoligonucleotides. In one embodiment, the isolation of a polymorphiclocus is accomplished by polymerase chain reaction amplification. In analternate embodiment, the polymerase chain reaction is conducted in thepresence of a nucleotide analog, including but not limited to the groupcomprising 7-deaza-dATP, 7-deaza-dGTP and dUTP. It is contemplated thatthe polymerase chain reaction amplification will employ oligonucleotideprimers matching or complementary to consensus gene sequences derivedfrom the polymorphic locus. In one embodiment, the polymorphic locuscomprises a ribosomal RNA gene. In a particularly preferred embodiment,the ribosomal RNA gene is a 16S ribosomal RNA gene.

The present invention also contemplates a process for creating a recordreference library of genetic fingerprints characteristic (i.e.,diagnostic) of one or more alleles of the various microorganisms,comprising the steps of providing a nucleic acid target derived frommicrobial gene sequences; comprising the steps of extracting nucleicacid from a sample suspected of containing one or more microorganisms;and contacting the extracted nucleic acid with one or moreoligonucleotide probes under conditions such that the extracted nucleicacid forms one or more secondary structures and interacts with one ormore probes; detecting the captured complexes; and maintaining atestable record reference of the captured complexes.

By the term “genetic fingerprint” it is meant that changes in thesequence of the nucleic acid (e.g., a deletion, insertion or a singlepoint substitution) alter both the sequences detectable by standard basepairing, and alter the structures formed, thus changing the profile ofinteractions between the target and the probe oligonucleotides (e.g.,altering the identity of the probes with which interaction occurs and/oraltering the site/s or strength of the interaction). The measure of theidentity of the probes bound and the strength of the interactionsconstitutes an informative profile that can serve as a “fingerprint” ofthe nucleic acid, reflecting the sequence and allowing rapid detectionand identification of variants.

The methods of the present invention allow for simultaneous analysis ofboth strands (e.g., the sense and antisense strands) and are ideal forhigh-level multiplexing. The products produced are amenable toqualitative, quantitative and positional analysis. The present methodsmay be automated and may be practiced in solution or in the solid phase(e.g., on a solid support). The present methods are powerful in thatthey allow for analysis of longer fragments of nucleic acid than currentmethodologies.

The present invention further provides methods for determination ofstructure formation in nucleic acid targets, comprising the steps of: a)providing: i) a folded target having a deoxyribonucleic acid sequencecomprising one or more double stranded regions, and one or more singlestranded regions, and further comprising two or more non-contiguousportions, and one or more intervening regions; and ii) one or morebridging oligonucleotide probes complementary to two or morenon-contiguous portions of the folded target; and b) mixing the foldedtarget and one or more bridging oligonucleotide probes under conditionssuch that the bridging oligonucleotide probes hybridize to the foldedtarget to form a probe/folded target complex.

In preferred embodiments, the one or more intervening regions of thefolded targets comprise at least five nucleotides. In yet otherembodiments, either of the targets and/or either of the bridgingoligonucleotides contain intervening regions comprised of non-nucleotidespacers of any length. In a preferred embodiment, the first and secondoligonucleotide probes comprise DNA. In alternative embodiments, themethod further comprises detecting the presence of the probe/foldedtarget complex. In yet other embodiments, the method further comprisesquantitating the amount of probe/folded target complex formed. In yetother embodiments of the method, the bridging oligonucleotide probe inthe probe/folded target complex is hybridized to at least one singlestranded region of the folded target.

The method is not limited by the nature of the target DNA employed toprovide the folded target DNA, nor is the method limited by the mannerin which the folded target DNA is generated. The method is also notlimited by the nature of the bridging oligonucleotide probes; theseprobes may comprise DNA, RNA, PNA and combinations thereof as well ascomprise modified nucleotides, universal bases, adducts, etc.

In a preferred embodiment, the method further comprises detecting thepresence of the probe/folded target complex. When a detection step isemployed either the bridging oligonucleotide probe or the target DNA (orboth) may comprise a label (i.e., a detectable moiety); the invention isnot limited by the nature of the label employed or the location of thelabel (i.e., 5′ end, 3′ end, internal to the DNA sequence). A widevariety of suitable labels are known to the art and include fluorescein,tetrachlorofluorescein, hexachlorofluorescein, Cy3, Cy5, digoxigenin,radioisotopes (e.g., ³²P, ³⁵S). In another preferred embodiment, themethod further comprises quantitating the amount of probe/folded targetcomplex formed. The method is not limited by the means used forquantification; when a labeled folded target DNA is employed (e.g.,fluorescein or ³²P), the art knows means for quantification (e.g.,determination of the amount of fluorescence or radioactivity present inthe probe/folded target complex).

Detection of the probe/folded target complex may also involve acatalyzed reaction on the probe that can only occur upon binding. It iscontemplated that such catalyzed reaction may be mediated by an enzyme.By way of example, but not by way of limitation, the bound bridgingoligonucleotide probe may be extended by a DNA polymerase, joined toanother nucleic acid by the action of a ligase, or cleaved by astructure-specific nuclease. It is further contemplated that thecatalytic action may be chemical, rather then enzymatic. For example,the cleavage of nucleic acid by compounds such as phenanthroline-Cu isspecific for duplexed structures. It is contemplated that any chemicalthat can act upon nucleic acid in a manner that is responsive to thestrandedness or other structural feature of the complex of the targetmay be used in the detection of the probe/folded target complex.

It is contemplated that any catalyzed reaction that is specificallyoperative on a duplex formed between a target nucleic acid and asubstantially complementary probe may be configured to perform on thebridging probe/folded target complex.

In another embodiment the bound probe may participate in a reactionrequiring a one or more additional nucleic acids, such as ligationreaction a polymerase chain reaction, a 5′ nuclease reaction, (Lyamichevet al., Science 260: 778 [1993]; U.S. Pat. No. 5,422,253, hereinincorporated by reference), or an Invader™ invasive cleavage reaction(PCT International Application No. PCT/US97/01072 [WO 97/27214];co-pending application Ser. Nos. 08/599,491, 08/682,853, 08/756,386,08/759,038, and 08/823,516, all of which are herein incorporated byreference). In one embodiment, the additional nucleic acid includesanother hybridized probe. In another embodiment, the additional nucleicacid included the target. In a preferred embodiment, the additionalnucleic acid includes a bridging oligonucleotide probe complementary totwo or more non-contiguous portions of the folded target.

It is contemplated that a nucleic acid on which the catalyzed reactionacts may be labeled. Thus detection of the complex on which thecatalyzed reaction has acted may comprise detection of a labeled productor products of that reaction. The invention is not limited by the natureof the label used, including, but not limited to, labels which comprisea dye or a radionuclide (e.g., ³²P), fluorescein moiety, a biotinmoiety, luminogenic, fluorogenic, phosphorescent, or fluorophores incombination with moieties that can suppress emission by fluorescenceresonance energy transfer (FRET). Numerous methods are available for thedetection of nucleic acids containing any of the above-listed labels.For example, biotin-labeled oligonucleotide(s) may be detected usingnon-isotopic detection methods which employ streptavidin-alkalinephosphatase conjugates. Fluorescein-labeled oligonucleotide(s) may bedetected using a fluorescein-imager. The oligonucleotides may be labeledwith different labels. The different labels may be present on the probebefore the catalytic reaction. In this embodiment the release of thelabels from attachment to the same complex (e.g., by FRET analysis), maybe used to detect formation of the probe/folded target complex.Alternatively, one or more of the labels may be added to the complex asa result of the catalytic reaction (e.g., by ligation to a labelednucleic acid or by polymerization using labeled nucleosidetriphosphates).

It is also contemplated that labeled oligonucleotides (reacted orunreacted) may be separated by means other than electrophoresis. Forexample, biotin-labeled oligonucleotides may be separated from nucleicacid present in the reaction mixture using para-magnetic or magneticbeads, or particles which are coated with avidin (or streptavidin). Inthis manner, the biotinylated oligonucleotide/avidin-magnetic beadcomplex can be physically separated from the other components in themixture by exposing the complexes to a magnetic field. Additionally, thesignal from the reacted oligonucleotides may be resolved from that ofthe unreacted oligonucleotides without physical separation. For example,a change in size as may be caused by binding to another oligonucleotide,or by cleavage, ligation or polymerase extension of at least one nucleicacid in the complex, will change the rate of rotation in solution,allowing of fluorescently labeled complexes or product molecules to bedetected by fluorescence polarization analysis. However, it is notintended that the means of analysis be limited to those methods of citedabove. Those skilled in the art of nucleic acid analysis will appreciatethat there are numerous additional methods for the analysis of both oflabeled and unlabeled nucleic acids that are readily adaptable for thedetection of the probe/folded target complexes of the present invention.

In another preferred embodiment, the bridging oligonucleotide probecomprises a bridging oligonucleotide having a moiety that permits itscapture by a solid support. The invention is not limited by the natureof the moiety employed to permit capture. Numerous suitable moieties areknown to the art, including but not limited to, biotin, avidin andstreptavidin. Further, it is known in the art that many small compounds,such as fluorescein and digoxigenin may serve as haptens for specificcapture by appropriate antibodies. Protein conjugates may also be usedto allow specific capture by antibodies.

In a preferred embodiment the detection of the presence of theprobe/folded target complex comprises exposing the probe/folded targetcomplex to a solid support under conditions such that the bridgingoligonucleotide probe is captured by the solid support. As discussed infurther detail below, numerous suitable solid supports are known to theart (e.g., beads, particles, dipsticks, wafers, chips, membranes or flatsurfaces composed of agarose, nylon, plastics such as polystyrenes,glass or silicon) and may be employed in the present methods.

In a particularly preferred embodiment, the moiety comprises a biotinmoiety and the solid support comprises a surface having a compoundcapable of binding to the biotin moiety, the compound selected from thegroup consisting of avidin and streptavidin.

In another embodiment, the folded target comprises a deoxyribonucleicacid sequence having a moiety that permits its capture by a solidsupport; as discussed above a number of suitable moieties are known andmay be employed in the present method. In yet another embodiment, thedetection of the presence of the probe/folded target complex comprisesexposing the probe/folded target complex to a solid support underconditions such that the folded target is captured by the solid support.In a preferred embodiment, the moiety comprises a biotin moiety and thesolid support comprises a surface having a compound capable of bindingto the biotin moiety, the compound selected from the group consisting ofavidin and streptavidin.

In a preferred embodiment, the bridging oligonucleotide probe isattached to a solid support; the probe is attached to the solid supportin such a manner that the bridging oligonucleotide probe is availablefor hybridization with the folded target nucleic acid. The invention isnot limited by the means employed to attach the bridging oligonucleotideprobe to the solid support. The bridging oligonucleotide probe may besynthesized in situ on the solid support or the probe may be attached(post-synthesis) to the solid support via a moiety present on thebridging oligonucleotide probe (e.g., using a biotinylated probe andsolid support comprising avidin or streptavidin). In another preferredembodiment, the folded target nucleic acid is attached to a solidsupport; this may be accomplished for example using a moiety present onthe folded target (e.g., using a biotinylated target nucleic acid andsolid support comprising avidin or streptavidin).

The present invention also provides methods for analyzing the structureof nucleic acid targets, comprising: a) providing: i) a first foldedtarget having a nucleic acid sequence comprising first and secondportions, the first and second portions each comprising one or moredouble stranded regions and one or more single stranded regions; ii) asecond folded target having a nucleic acid sequence comprising a firstportion that is identical to the first portion of the first foldedtarget and a second portion that differs from the second portion of thefirst folded target because of a variation in nucleic acid sequencerelative to the first folded target, the first and second portions eachcomprising one or more double stranded regions and one or more singlestranded regions; iii) first and second bridging oligonucleotides,wherein the first bridging oligonucleotide is complementary to the firstportion of the first and second folded targets and the second bridgingoligonucleotide is complementary to the second portion of the first andsecond folded targets; and iv) a solid support comprising first, second,third and fourth testing zones, each zone capable of capturing andimmobilizing the first and second bridging oligonucleotides; b)contacting the first folded target with the first bridgingoligonucleotide under conditions such that the first bridgingoligonucleotide binds to the first folded target to form a probe/foldedtarget complex in a first mixture; c) contacting the first folded targetwith the second bridging oligonucleotide under conditions such that thesecond bridging oligonucleotide binds to the first folded target to forma probe/folded target complex in a second mixture; d) contacting thesecond folded target with the first bridging oligonucleotide to form athird mixture; e) contacting the second folded target with the secondbridging oligonucleotide to form fourth mixture; and f) adding thefirst, second, third and fourth mixtures to the first, second, third andfourth testing zones of the solid support, respectively, underconditions such that the first and second bridging oligonucleotides arecaptured and immobilized.

The method is not limited by the nature of the first and second targets.The first and/or second target may comprise one or more non-contiguousregions, as well as one or more intervening regions. In preferredembodiments, the intervening regions comprise at least five nucleotides.The method is also not limited by the nature of the bridgingoligonucleotide probes; these bridging oligonucleotide probes maycomprise DNA, RNA, PNA and combinations thereof as well as comprisemodified nucleotides, universal bases, adducts, etc. In someembodiments, the first and/or second bridging oligonucleotide probescomprise one or more intervening regions. In alternative embodiments,the intervening region of the bridging oligonucleotide probes comprisesat least two nucleotides. In yet other embodiments, either of thetargets and/or either of the bridging oligonucleotides containintervening regions comprised of non-nucleotide spacers of any length.In a preferred embodiment, the first and second oligonucleotide probescomprise DNA. In a preferred embodiment, the first and second bridgingoligonucleotide probes comprise DNA.

In alternative embodiments, the first bridging oligonucleotide in stepd) does not substantially hybridize to the second folded target. In yetanother embodiment, the hybridization of the first bridgingoligonucleotide in step d) to the second folded target is reducedrelative to the hybridization of the first bridging oligonucleotide instep c) to the first folded target. In further embodiments, the firstand second targets comprise DNA, and/or the first and second bridgingoligonucleotides comprise DNA.

The present invention also provides methods for analyzing folded nucleicacid targets, comprising: a) providing: i) a first folded target havinga nucleic acid sequence comprising first and second portions, whereinthe first and second portions each comprise one or more double strandedregions and one or more single stranded regions; ii) a second foldedtarget having a nucleic acid sequence comprising a first portion that isidentical to the first portion of the first folded target, and a secondportion that differs from the second portion of the first folded targetbecause of a variation in nucleic acid sequence relative to the firstfolded target, the first and second portions each comprising one or moredouble stranded regions and one or more single stranded regions; iii) asolid support comprising first and second testing zones, each of thezones comprising immobilized first and second bridging oligonucleotides,the first bridging oligonucleotide being complementary to the firstportion of the first and second folded targets and second bridgingoligonucleotide being complementary to the second portion of the firstand second folded targets; and b) contacting the first and second foldedtargets with the solid support under conditions such that the first andsecond bridging oligonucleotides hybridize to the first folded target toform a probe/folded target complex.

In some embodiments, the contacting of step b) comprises adding thefirst folded target to the first testing zone and adding the secondfolded target to the second testing zone. In alternative embodiments,the first and second bridging oligonucleotides are immobilized inseparate portions of the testing zones. In yet other embodiments, thefirst bridging oligonucleotide in the second testing zone does notsubstantially hybridize to the second folded target. In furtherembodiments, the first bridging oligonucleotide in the second testingzone hybridizes to the second folded target with a reduced efficiencycompared to the hybridization of the first bridging oligonucleotide infirst testing zone to the first folded target. The method is not limitedby the nature of, nor the method of generating the first and secondfolded targets. The method is also not limited by the nature of, or themethod of generating the oligonucleotide probes; these probes maycomprise DNA, RNA, PNA and combinations thereof as well as comprisemodified nucleotides, universal bases, adducts, etc. In someembodiments, the first and/or second folded target comprises one or moreintervening region comprised of at least five nucleotides. In yet otherembodiments, the first and/or second bridging oligonucleotide probecomprises one or more intervening regions comprised of at least twonucleotides. In yet other embodiments, either of the targets and/oreither of the bridging oligonucleotides contain intervening regionscomprised of non-nucleotide spacers of any length. In a preferredembodiment, the first and second oligonucleotide probes comprise DNA.The invention is not limited by the nature of the solid support employedas discussed above. In some preferred embodiments of the method, thefirst and second folded targets comprise DNA. In alternativeembodiments, the first and second folded targets comprise RNA. In yetother embodiments, the first and second bridging oligonucleotidescomprise DNA.

The present invention provides methods for detection of structurednucleic acid targets, comprising the steps of: a) providing: i) a foldedtarget having a nucleic acid sequence comprising one or more doublestranded regions, and one or more single stranded regions, and furthercomprising two or more non-contiguous portions, and one or moreintervening regions; ii) at least one bridging oligonucleotide probecapable of binding to two or more non-contiguous portions of said foldedtarget; and iii) a reactant; b) mixing said folded target and said probeunder conditions such that said probe hybridizes to said folded targetto form a probe/folded target complex; and c) treating said probe/foldedtarget complex with said reactant to produce at least one modifiedprobe. In one embodiment the method further provides for the detectionof said modified probe.

The present invention further provides a method, comprising: a)providing target nucleic acid comprising first and second non-contiguoussingle-stranded regions separated by an intervening region comprising adouble-stranded portion; a bridging oligonucleotide capable of bindingto said first and second non-contiguous single-stranded regions; and areactant selected from the group consisting of polymerases and ligases;and mixing said target nucleic acid, said bridging oligonucleotide andsaid reactant under conditions such that said bridging oligonucleotideis modified to produce a modified oligonucleotide.

In some embodiments of the methods, the reactant is a polymerase, whilein yet other embodiments, the modified oligonucleotide comprises anextended oligonucleotide. In still other embodiments, the reactant is apolymerase and the modified oligonucleotide comprises extendedoligonucleotide. In yet other embodiments, the reactant is a ligase,while in yet other embodiments, the modified oligonucleotide comprises aligated oligonucleotide. In still other embodiments, the reactant is aligase and the modified oligonucleotide comprises a ligatedoligonucleotide.

In yet other embodiments of the method, the bridging oligonucleotide iscapable of binding to fewer than ten nucleotides of each of said firstand second non-contiguous single-stranded regions. In still otherembodiments, the bridging oligonucleotide is capable of binding to eightor fewer nucleotides of each of said first and second non-contiguoussingle-stranded regions.

In further embodiments of the method the target nucleic acid is DNA,while in some preferred embodiments, the DNA is viral DNA. In yet otherpreferred embodiments, the virus is selected from the group consistingof Parvoviridae, Papovaviridae, Adenoviridae, Hepadnaviridae,Herpesvindae, Iridoviridae, and Poxviridae. For example, it is intendedthat the present invention encompass methods for the detection of anyDNA-containing virus, including, but not limited to parvoviruses,dependoviruses, papillomaviruses, polyomaviruses, mastadenoviruses,aviadenoviruses, hepadnaviruses, simplexviruses [such as herpes simplexvirus 1 and 2], varicelloviruses, cytomegaloviruses, muromegaloviruses,lymphocryptoviruses, thetalymphocryptoviruses, rhadinoviruses,iridoviruses, ranaviruses, pisciniviruses, orthopoxviruses,parapoxviruses, avipoxviruses, capripoxviruses, leporipoxviruses,suipoxviruses, yatapoxviruses, and mulluscivirus). Thus, it is notintended that the present invention be limited to any DNA virus family.

In further embodiments of the method the target nucleic acid is RNA,while in some preferred embodiments, the RNA is viral RNA. In yet otherpreferred embodiments, the virus is selected from the group consistingof Picornaviridae, Caliciviridae, Reoviridae, Togaviridae, Flaviviridae,Orthomyxoviridae, Paramyxoviridae, Arenaviridae, Rhabdoviridae,Coronaviridae, Bunyaviridae, and Retroviridae. For example, it isintended that the present invention encompass methods for the detectionof RNA-containing virus, including, but not limited to enteroviruses(e.g., polioviruses, Coxsackieviruses, echoviruses, enteroviruses,hepatitis A virus, encephalomyocarditis virus, mengovirus, rhinoviruses,and aphthoviruses), caliciviruses, reoviruses, orbiviruses, rotaviruses,birnaviruses, alphaviruses, rubiviruses, pestiviruses, flaviviruses(e.g., hepatitis C virus, yellow fever viruses, dengue, Japanese, MurrayValley, and St. Louis encephalitis viruses, West Nile fever virus,Kyanasur Forest disease virus, Omsk hemorrhagic fever virus, Europeanand Far Eastern tick-borne encephalitis viruses, and louping ill virus),influenzaviruses (e.g, types A, B, and C), paramyxoviruses,morbilliviruses, pneumoviruses, veisculoviruses, lyssaviruses,filoviruses, coronaviruses, bunyaviruses, phleboviruses, nairoviruses,uukuviruses, hantaviruses, sarcoma and leukemia viruses, oncoviruses,HTLV, spumaviruses, lentiviruses, and arenaviruses).

The present invention also provides a method, comprising: a) providingtarget nucleic acid comprising first and second non-contiguoussingle-stranded regions separated by an intervening region comprising adouble-stranded region; a bridging oligonucleotide capable of binding tosaid first and second non-contiguous single-stranded regions; a secondoligonucleotide capable of binding to a portion of said firstnon-contiguous single-stranded region; and a cleavage means; b) mixingsaid target nucleic acid, said bridging oligonucleotide, said secondoligonucleotide, and said cleavage means under conditions such thateither said second oligonucleotide or said bridging oligonucleotide iscleaved.

In some preferred embodiments, the cleavage means comprises a nuclease.In other preferred embodiments, the cleavage means comprises athermostable 5′ nuclease. In still other preferred embodiments, thethermostable 540 nuclease comprises an altered polymerase derived from anative polymerases of Thermus species.

In other embodiments of the method, the conditions of mixing allow forhybridization of said bridging oligonucleotide and said secondoligonucleotide to said target nucleic acid so as to define a region ofoverlap of said oligonucleotides. In some embodiments, the region ofoverlap comprises one base, while in other embodiments, the region ofoverlap comprises more than one base.

In further embodiments of the method the target nucleic acid is DNA,while in some preferred embodiments, the DNA is viral DNA. In yet otherpreferred embodiments, the virus is selected from the group consistingof Parvoviridae, Papovaviridae, Adenoviridae, Hepadnaviridae,Herpesviridae, Iridoviridae, and Poxviridae. For example, it is intendedthat the present invention encompass methods for the detection of anyDNA-containing virus, including, but not limited to parvoviruses,dependoviruses, papillomaviruses, polyomaviruses, mastadenoviruses,aviadenoviruses, hepadnaviruses, simplexviruses [such as herpes simplexvirus 1 and 2], varicelloviruses, cytomegaloviruses, muromegaloviruses,lymphocryptoviruses, thetalymphocryptoviruses, rhadinoviruses,iridoviruses, ranaviruses, pisciniviruses, orthopoxviruses,parapoxviruses, avipoxviruses, capripoxviruses, leporipoxviruses,suipoxviruses, yatapoxviruses, and mulluscipoxvirus). Thus, it is notintended that the present invention be limited to any DNA virus family.

In further embodiments of the method the target nucleic acid is RNA,while in some preferred embodiments, the RNA is viral RNA. In yet otherpreferred embodiments, the virus is selected from the group consistingof Picornaviridae, Caliciviridae, Reoviridae, Togaviridae, Flaviviridae,Orthomyxoviridae, Paramyxoviridae, Arenaviridae, Rhabdoviridae,Coronaviridae, Bunyaviridae, and Retroviridae. For example, it isintended that the present invention encompass methods for the detectionof RNA-containing virus, including, but not limited to enteroviruses(e.g., polioviruses, Coxsackieviruses, echoviruses, enteroviruses,hepatitis A virus, encephalomyocarditis virus, mengovirus, rhinoviruses,and aphthoviruses), caliciviruses, reoviruses, orbiviruses, rotaviruses,birnaviruses, alphaviruses, rubiviruses, pestiviruses, flaviviruses([e.g., hepatitis C virus, yellow fever viruses, dengue, Japanese,Murray Valley, and St. Louis encephalitis viruses, West Nile fevervirus, Kyanasur Forest disease virus, Omsk hemorrhagic fever virus,European and Far Eastern tick-borne encephalitis viruses, and loupingill virus], influenzaviruses (e.g, types A, B, and C), paramyxoviruses,morbilliviruses, pneumoviruses, veisculoviruses, lyssaviruses,filoviruses, coronaviruses, bunyaviruses, phleboviruses, nairoviruses,uukuviruses, hantaviruses, sarcoma and leukemia viruses, oncoviruses,HTLV, spumaviruses, lentiviruses, and arenaviruses).

The present invention also provides a method, comprising: a) providingtarget nucleic acid comprising first and second non-contiguoussingle-stranded regions separated by an intervening region, saidintervening region comprising a first double-stranded portion and asecond double-stranded portion separated by a connecting single-strandedportion; and a bridging oligonucleotide capable of binding to said firstand second non-contiguous single-stranded regions; and b) mixing saidtarget nucleic acid and said bridging oligonucleotide under conditionssuch that said bridging oligonucleotide hybridizes to said target toform an oligonucleotide/target complex.

In further embodiments of the method the target nucleic acid is DNA,while in some preferred embodiments, the DNA is viral DNA. In yet otherpreferred embodiments, the virus is selected from the group consistingof Parvoviridae, Papovaviridae, Adenoviridae, Hepadnaviridae,Herpesviridae, Iridoviridae, and Poxviridae. For example, it is intendedthat the present invention encompass methods for the detection of anyDNA-containing virus, including, but not limited to parvoviruses,dependoviruses, papillomaviruses, polyomaviruses, mastadenoviruses,aviadenoviruses, hepadnaviruses, simplexviruses [such as herpes simplexvirus 1 and 2], varicelloviruses, cytomegaloviruses, muromegaloviruses,lymphocryptoviruses, thetalymphocryptoviruses, rhadinoviruses,iridoviruses, ranaviruses, pisciniviruses, orthopoxviruses,parapoxviruses, avipoxviruses, capripoxviruses, leporipoxviruses,suipoxviruses, yatapoxviruses, and mulluscipoxvirus). Thus, it is notintended that the present invention be limited to any DNA virus family.

In further embodiments of the method the target nucleic acid is RNA,while in some preferred embodiments, the RNA is viral RNA. In yet otherpreferred embodiments, the virus is selected from the group consistingof Picornaviridae, Caliciviridae, Reoviridae, Togaviridae, Flaviviridae,Orthomyxoviridae, Paramyxoviridae, Arenaviridae, Rhabdoviridae,Coronaviridae, Bunyaviridae, and Retroviridae. For example, it isintended that the present invention encompass methods for the detectionof RNA-containing virus, including, but not limited to enteroviruses(e.g., polioviruses, Coxsackieviruses, echoviruses, enteroviruses,hepatitis A virus, encephalomyocarditis virus, mengovirus, rhinoviruses,and aphthoviruses), caliciviruses, reoviruses, orbiviruses, rotaviruses,birnaviruses, alphaviruses, rubiviruses, pestiviruses, flaviviruses(e.g., hepatitis C virus, yellow fever viruses, dengue, Japanese, MurrayValley, and St. Louis encephalitis viruses, West Nile fever virus,Kyanasur Forest disease virus, Omsk hemorrhagic fever virus, Europeanand Far Eastern tick-borne encephalitis viruses, and louping ill virus),influenzaviruses (e.g, types A, B, and C), paramyxoviruses,morbilliviruses, pneumoviruses, veisculoviruses, lyssaviruses,filoviruses, coronaviruses, bunyaviruses, phleboviruses, nairoviruses,uukuviruses, hantaviruses, sarcoma and leukemia viruses, oncoviruses,HTLV, spumaviruses, lentiviruses, and arenaviruses).

The present invention further provides a method for the analysis ofnucleic acid structures comprising; providing a sequence data inputmeans (defined as any means [e.g., a computer input device and softwarefor receiving and storing the sequence information] for entering nucleicacid sequence information into a device capable of storing and/orprocessing the data), a cleavage data input means (defined as any means[e.g., a computer input device and software for receiving and storingthe sequence information] for entering information regarding thelocation of a cleavage site in a nucleic acid into a device capable ofstoring and/or processing the data), and a nucleic acid structureprediction means (defined as any means [e.g., software designed topredict the structure of nucleic acids or proteins based on sequencedata and other data inputs] capable of predicting nucleic acid sequencebased on input data); providing nucleic acid sequence data (defined asany data relating to the sequence of one or more nucleic acidcompositions) to said sequence data input means to produce sequence dataresults; providing structure-specific cleavage data (defined as any datarelating to the cleavage status of one or more nucleic acidcompositions) to said cleavage data input means to produce cleavage dataresults; and providing said sequence data results and said cleavage dataresults to said nucleic acid structure prediction means to produce apredicted nucleic acid structure (defined as any structure capable ofinterpretation by users [e.g., a pictographic display] or by a devicecapable of relaying the structural information to a user in anyinterpretable form).

In some embodiments, the present invention further provides methods forthe analysis of nucleic acid structures comprising the steps of e)providing a basepair data input means and a second nucleic acidstructure prediction means; f) providing basepair data to said basepairdata input means to produce basepair data results; and g) providing saidsequence data results, said cleavage data results, and said basepairdata results to said second nucleic acid structure prediction means toproduce a second predicted nucleic acid structure.

DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic of one embodiment of the detection methodsof the present invention.

FIGS. 2A-2D provide schematic representations of a segment of the katGgene from M. tuberculosis. Depending on the sequence, the segment of theDNA can form the stem-loop structures depicted in 2C and 2D. The arrowsin 2C and 2D show the sites that are cleaved when these structures aretreated by the structure specific Cleavase® I nuclease. The black bar tothe left of each structure indicates the region to which the katG probewould bind, with the pointed kink in the bar indicating a site ofmismatch between the probe and the katG target. FIG. 2A shows apolynucleotide that spans the region from residues 29 to 391 of SEQ IDNO:1. FIG. 2B shows a polynucleotide that spans the region from residues29 to 391 of SEQ ID NO:2. FIG. 2C shows a polynucleotide that spans theregion from residues 29 to 391 of SEQ ID NO:3. FIG. 2D shows apolynucleotide that spans the region from residues 29 to 391 of SEQ IDNO:4.

FIG. 3 shows at left a fluorescence imager scan of the cleavage patternsgenerated using the CFLP® method on the katG substrates. The lettersabove the lanes indicate that these DNA fragments contain to thecorresponding structures diagramed in FIGS. 2A-2D. An arrow indicatesthe 37 nucleotide (nt) product of cleavage at the site indicated by thearrows in FIGS. 2C-2D. The graph at the right depicts the fluorescenceintensity measured when each of the molecules depicted in FIGS. 2A-2Dwas complexed to the katG capture probe and bound to a solid support ina structure probing assay.

FIG. 4 show a graph that depicts the fluorescence intensity measuredwhen two variants of the katG target DNA with different amounts offlanking sequence were bound to a microtiter plate using a singlecapture probe.

FIG. 5 shows an analysis of several types of HCV by both the CFLP®method and by DNA sequencing. The sequence lanes were resolved besidethe lanes showing the products of CFLP® cleavage. This allowed preciseidentification of the sites cleaved, and therefore the regions ofstructure, in the analysis of each of the HCV genotypes. The probesselected to interact in these regions are indicated to the right (SEQ IDNOS:11-19).

FIG. 6 provides an alignment of sequences that have been determined forthe HCV genotypes examined in Example 3. The sites within the HCVtargets which the probes have been designed to complement are underlinedand shown in bold. The numbers of the probes are indicated above eachsite. SEQ ID NOS:20-23 are shown in FIG. 6.

FIG. 7 shows four graphs depicting the fluorescence signal measuredafter the solid support capture of the indicated HCV types by theindicated probes.

FIGS. 8A, B and C show graphs depicting the fluorescence signal measuredafter the solid support capture of the indicated HCV types by theindicated probes, at temperatures ranging from room temperature(approximately 22° C.) to 50° C.

FIGS. 9A-9D show graphs depicting the fluorescence signal measured afterthe solid support capture of different HCV types from clinical samples,by the indicated probes.

FIG. 10 shows schematic representations of the folded structures thatwould be assumed by each of the three test molecules, #80 (SEQ IDNO:39), #81 (SEQ ID NO:40) and #82 (SEQ ID NO:41).

FIGS. 11A and 11B show schematic representations of the captureoligonucleotides used in these studies. While are were tested with allthree of the test molecules depicted in FIG. 10, for convenience theyare shown aligned with their complementary regions in test molecule #80(SEQ ID NO:39). FIG. 11A shows probe molecules Nos. 2, FD91, 80, 78, 4,79, and 116-188 which correspond, respectively, to SEQ ID NOS: 51, 50,39, 42, 43, 44, 47, 48, and 49. FIG. 11B shows probe molecules Nos: 79,114, and 115, which correspond, respectively, to SEQ ID NOS:44-46.

FIGS. 12A-12D show graphs depicting the fluorescence signal measuredafter the solid support capture of the three test molecules, #80 (SEQ IDNO:39), #81 (SEQ ID NO:40), and #82 (SEQ ID NO:41) by the indicatedprobes. The wider fourth panel (FIG. 12D), shows the fluorescence signalfrom each of the first three panels re-drawn together on a single scaleof fluorescence intensity, for ease of comparison.

FIGS. 13A and 13B show graphs depicting the fluorescence signal measuredafter the solid support capture of the three test molecules, #80 (SEQ IDNO:39), #81 (SEQ ID NO:40), and #82 (SEQ ID NO:41) by the indicatedprobes. The names of the probes used in each capture test are indicatedabove each individual panel in these Figure panels.

FIG. 14 shows a schematic diagram of the process for selecting twosegments of bridging oligonucleotide based on the data from the use of5′ and 3′ nucleases to cleave a folded structure. Such cleavagereactions can be used to locate regions that are either upstream anddownstream of folded structures, facilitating selection of complementarysequences to compose bridging oligonucleotides.

FIG. 15 shows an alignment of four 244 nt segments of HCV, representingtypes 1a (SEQ ID NO:124), 1b (SEQ ID NO:125), 2a/c (SEQ ID NO:126) and3a (SEQ ID NO: 127). Type la is shown in its entirety, while only thedifferences are indicated for the other types. Cleavage sites generatedby CFLP® cleavage are indicated by vertical lines along the sequence,with the weakest cleavage sites shown as broken lines.

FIGS. 16A and 16B show schematic diagrams of two possible secondarystructures for a 244 nt fragment (SEQ ID NO:128) derived from HCV type1a.

FIG. 17B shows schematic diagrams of two of the predicted structures fora region from residue 70 to 128 of the 244 nt amplicon derived from HCVtype 1a (SEQ ID NO: 128). The CFLP® data indicates that the target DNAassumes multiple conformations in solution, each contributing to thecleavage pattern (Brow et al., supra).

FIG. 17C shows schematic diagram of three bridging oligonucleotidesdesigned two interact with the predicted structures (residue 84 to 213,and residue 110 to 204, respectively of SEQ ID NO:128) for this region“b” “m” and “n” (SEQ ID NOS:53, 64, and 65). The regions that arecomplementary as aligned to the target are indicated by a black linebetween the strands.

FIGS. 18A-D show schematic diagrams of the predicted structures for aregion of the 244 nt amplicon derived from HCV types la (residues 70 to213 of SEQ ID NO:124), 1b (residues 46 to 213 of SEQ ID NO:125), 2a/c(residues 77 to 213 of SEQ ID NO:126) and 3a (residues 77 to 213 of SEQID NO:127), respectively. In FIGS. 18 B-D the bases that differ from thetype 1a sequence are shown in bold. Each is aligned with bridgingoligonucleotides “b,” “i,” “j,” “k,” “c,” and “d” of six differentdesigns (SEQ ID NOS:53, 54, 55, 56, 57, and 58). The regions that arecomplementary as aligned to the target are indicated by a black linebetween the strands. The 3′ terminal contact sequence of each probe(excepting “c”) is complementary to eight contiguous target basesupstream of the right most stem, but representation of the small centralstem prevents showing this alignment.

FIGS. 19 shows graphs depicting the fluorescence signal measured afterthe solid support capture of the amplicons derived from HCV types 1a,1b, 2a/c and 3a by the indicated probes. The letters identifying theprobes used in each capture test are indicated below each bar, and thesignal in arbitrary fluorescence units is shown on the left of eachpanel.

FIG. 20A shows a schematic diagram of a structure in the ampliconderived from HCV type 1a (residues 136 to 213 of SEQ ID NO:124) alignedwith non-bridging probes “a” (SEQ ID NO:52) and “e” (SEQ ID NO:59) andbridging probes “b”-“d” (SEQ ID NOs: 53, 57 and 58, respectively). Theregions that are complementary as aligned to the target are indicated bya black line between the strands.

FIG. 20B shows a schematic diagram of a structure in the ampliconderived from HCV type 1a (residues 136 to 213 of SEQ ID NO:124) as itmight be expected to pair with the fully complementary non-bridgingoligonucleotide “a” (SEQ ID NO:52). The regions that are complementaryas aligned to the target are indicated by a black line between thestrands.

FIG. 21 shows a fluorescence imager scan of the products of primerextension reactions using the probes depicted in FIG. 20A and the foldedtarget strands derived from HCV types 1a, 1b, 2a/c and 3a, or usinghuman genomic DNA as a control, as indicated above each lane. An arrowindicates the 170 nucleotide (nt) product of extension.

FIG. 22 shows a schematic diagram of a structure in the amplicon derivedfrom HCV type 1a (residues 136 to 213 of SEQ ID NO:124) aligned withnon-bridging probes “a” and “e” and bridging probe “b” (SEQ ID NOS:52,53, and 59, respectively). The regions that are complementary as alignedto the target are indicated by a black line between the strands.

FIG. 23 shows a fluorescence imager scan of the products of primerextension reactions using the probes and target depicted in FIG. 22 inreactions performed over a range of temperatures. The temperatures ofeach reaction are indicated at the top of the panel, and the unreactedprobes are indicated by arrows and their letters on the left. An arrowindicates the 170 nucleotide (nt) product of extension.

FIG. 24 shows a schematic diagram of a structure in the amplicon derivedfrom HCV type 1a (residues 136 to 213 of SEQ ID NO:124) aligned withnon-bridging probes “a” and “e” and bridging probes “b”-“d” and ligationoligonucleotide “f” (SEQ ID NOS:52, 59, 53, 57, 58, and 62,respectively). The regions that are complementary as aligned to thetarget are indicated by a black line between the strands.

FIGS. 25 shows graphs depicting the fluorescence signal measured afterthe solid support capture of the amplicons derived from HCV types 1a,1b, 2a/c and 3a by the indicated probes and combinations of probes. Theletters identifying the probes used in each capture test are indicatedbelow each bar, and the signal in arbitrary fluorescence units is shownon the left of each panel.

FIG. 26 shows a schematic diagram of an unstructured synthetic target“S.T.” (SEQ ID NO:63) aligned with non-bridging probes “a” and “e” andbridging probes “b”-“d” and ligation oligonucleotide “f” (SEQ ID NOS:52,59, 53, 57, 58, and 62, respectively). The regions that arecomplementary as aligned to the target are indicated by a black linebetween the strands.

FIG. 27 shows a fluorescence imager scan of the products of ligationreactions using the probes and targets depicted in FIGS. 24 and 26. Theunreacted probes are indicated at 8 and 18 nt by arrows on the left.Arrows indicates the 33 nt product of ligation between the probe “f” and“a”, “b”, “c” or “d”, and the 23 nt product of ligation between “f” and“e”.

FIG. 28 shows a fluorescence imager scan of the products of ligationreactions using the ligation probe “f” and the bridging probe “b” inreactions performed at various temperatures, using target ampliconsderived from HCV types 1a, 1b, 2a/c and 3a. Arrows on the left indicatethe unreacted probe at 18 nt the product of ligation at 33 nt.

FIGS. 29A and 29B show schematic diagrams of either a structure in theamplicon derived from HCV type 1a (residues 136 to 213 of SEQ IDNO:124), or an unstructured synthetic target “S.T.” (SEQ ID NO:63)respectively, aligned with non-bridging probes “a” and “e”, bridgingprobes “b”-“d” and invasive cleavage probe “g” (SEQ ID NOS: 52, 58, 59,53, 57, and 60, respectively). The regions that are complementary asaligned to the targets are indicated by a black line between thestrands.

FIG. 30 shows a fluorescence imager scan of the products of invasivecleavage reactions using the probes and targets depicted in FIGS. 29Aand 29B. The identities of the target DNA and probes used in eachreaction (in addition to the cleavage probe “g”; SEQ ID NO:60) areindicted above each lane, and the unreacted probes are indicated byarrows and their letters on the left. An arrow indicates the 4nucleotide (nt) product of cleavage.

FIG. 31 shows a schematic diagram of a structure in the amplicon derivedfrom HCV type 1a (residues 136 to 213 of SEQ ID NO:124) aligned withbridging probe “b” (SEQ ID NO:53) and invasive cleavage probe “h” (SEQID NO:61). The regions that are complementary as aligned to the targetare indicated by a black line between the strands.

FIG. 32 shows a fluorescence imager scan of the products of invasivecleavage reactions using the probes and target depicted in FIG. 31, inreactions performed over a range of temperatures, as indicated above thelanes. The identities of the target DNA and probes used in each reaction(in addition to the cleavage probe “h”; SEQ ID NO:61) are indicted aboveeach lane, and the unreacted probes are indicated by arrows and theirletters on the right. An arrow indicates the 4 nucleotide (nt) productof cleavage.

FIG. 33 shows a fluorescence imager scan of the products of invasivecleavage reactions using the probes and targets depicted in 29A and 31.The identities of the target DNA and probes used in each reaction areindicted above each lane, and the cleavage probes used ate indicatedbelow the lanes. The unreacted probes are indicated by arrows and theirletters on either side and arrows indicate the 4 nucleotide (nt) productof cleavage.

FIG. 34 is a schematic diagram showing one example of the use ofbridging oligonucleotides as primers in a polymerase chain reaction. The“a-e” designations in this Figure are used to indicate the general stepsin the reaction.

FIG. 35 is a schematic diagram showing two examples of target-dependentligation of bridging oligonucleotides, with subsequent detection of thebridged ligation product by a ligase chain reaction. The “a-c”designations in this Figure are used to indicate the steps in thereaction, with either step a or b being followed by step c (i.e., b doesnot follow a in the progression of the steps).

FIG. 36 shows a fluorescence imager scan of the cleavage patternsgenerated using the CFLP® method on a 128 nucleotide fragment derivedfrom the rpoB gene of M tuberculosis (right lane). A marker havingfragments of the indicated sizes (in nucleotides) is shown in the leftlane and the sizes of the significant cleavage bands from the rpoBfragment are indicated to the right of the panel.

FIG. 37A shows two schematic diagrams of two possible secondarystructures for a 128 nucleotide fragment (SEQ ID NO:72) derived from therpoB gene of M. tuberculosis.

FIG. 37B shows four schematic diagrams; one is of the stem predicted tofold when nucleotide 62 of the rpoB amplicon is basepaired to nucleotide114 (residues 54 to 122 of SEQ ID NO:72); three variant molecules,indicated as 1 (SEQ ID NO:88), 2 (SEQ ID NO:90), and 3 (SEQ ID NO:92),are also depicted.

FIG. 37C shows a schematic diagram of a structured site in the amplicon(residues 54 to 122 of SEQ ID NO:72) derived from the rpoB gene of M.tuberculosis having a basepair between nucleotides 62 and 114, alignedwith bridging probes having different spacer regions (SEQ ID NOS:106,105, 107, 108, and 109, respectively). The regions of the target thatare complementary to the probes are indicated by a black line below thetarget structure. A graph depicts the fluorescence signal measured afterthe solid support capture of this amplicon by the indicated probes. Thenumbers identifying the probes used in each capture test are indicatedabove each bar and the spacer in each probe is indicated below each bar.The fluorescence signal is shown on the left of the panel as apercentage of the signal measured in experiments using a linear(non-bridging) control probe for capture of this target.

FIG. 38A shows schematic diagrams of a three structured sites in theamplicon derived from the rpoB gene of M. tuberculosis aligned withbridging probes 17-20 (SEQ ID NOS:110, 111, 112, and 113). Inparticular, the top left structure represents residues 45 to 126 of SEQID NO:72 and the alignment of bridging probe SEQ ID NO:110. The Topright structure represents residues 61 to 118 of SEQ ID NO:72 and thealignment of bridging probe SEQ ID NO:111. The bottom left structurerepresents residues 67 to 128 of SEQ ID NO:72 and the alignment ofbridging probes SEQ ID NOS: 112 (top) and SEQ ID NO:113 (bottom),respectively. The regions that are complementary as aligned to thetarget are indicated by a black line between the strands. A graphdepicts the fluorescence signal measured after the solid support captureof these amplicons by the indicated probes. The numbers identifying theprobes used in each capture test are indicated below each bar, and thefluorescence signal is shown on the left of the panel as a percentage ofthe signal measured in experiments using a linear (non-bridging) controlprobe for capture of these targets.

FIG. 38B shows schematic diagrams of two structured sites, residues 70to 114, and residues 5 to 95 of SEQ ID NO:72, respectively, in theamplicon derived from the rpoB gene of M. tuberculosis aligned withbridging probes 78-106 and 63-87 (SEQ ID NOs:114 and 115, respectively).The regions that are complementary as aligned to the target areindicated by a black line between the strands. A graph depicts thefluorescence signal measured after the solid support capture of thisamplicon by the indicated probe. The numbers identifying the probes usedin each capture test are indicated below each bar, and the fluorescencesignal is shown on the left of the panel as a percentage of the signalmeasured in experiments using a linear (non-bridging) control probe forcapture of this target.

FIG. 38C shows schematic diagrams of three structured sites in theamplicon derived from the rpoB gene of M. tuberculosis, residues 76 to110, residues 49 to 119, and residues 54 to 122 of SEQ ID NO:72,respectively, aligned with bridging probes 84-102, 57-119 or 84-102 (SEQID NOs:116, 117, and 118, respectively). The regions that arecomplementary as aligned to the target are indicated by a black linebetween the strands. A graph depicts the fluorescence signal measuredafter the solid support capture of this amplicon by the indicated probe.The numbers identifying the probes used in each capture test areindicated below each bar, and the fluorescence signal is shown on theleft of the panel as a percentage of the signal measured in experimentsusing a linear (non-bridging) control probe for capture of this target.

FIG. 39 shows schematic diagrams of three possible structures (“a”residues 54 to 122 of SEQ ID NO:72), “b” residues 54 to 121 of SEQ IDNO:72), and “c” residues 55 to 95 of SEQ ID NO:72)) formed by theamplicon derived from the rpoB gene of M. tuberculosis. Each of thesethree structures could cause CFLP® cleavage 62 to 63 nucleotides fromthe 5′ end of this fragment, contributing signal in this region of theCFLP® gel pattern.

FIG. 40 shows a schematic diagram of structure “b” from FIG. 39(residues 54 to 121 of SEQ ID NO:72) aligned with a bridging probe (SEQID NO:118) that could create a four-way junction. A graph depicts thefluorescence signal measured after the solid support capture of twodifferent sized amplicons by this probe. The fluorescence signal isshown on the left of the panel as a percentage of the signal measured inexperiments using a linear (non-bridging) control probe for capture ofthese targets.

FIG. 41 shows schematic diagrams of structure “b” from FIG. 39, eitherunaltered (residues 54 to 121 of SEQ ID NO:72), or truncated and mutated(residues 54 to 113 of SEQ ID NO:92) to destabilize the shorter stem.Also depicted is bridging probe 62-98 (SEQ ID NO:119), designed tohybridize across the longer remaining stem, and a graph depicting thefluorescence signal measured after the solid support capture of theshortened amplicon by the indicated probe. The fluorescence signal isshown on the left of the panel as a percentage of the signal measured inexperiments using a linear (non-bridging) control probe for capture ofthis target.

FIG. 42 shows a schematic diagram of structure “c” from FIG. 39(residues 55 to 95 of SEQ ID NO:72) aligned with bridging probe 63-87(SEQ ID NO:115), and a graph depicting the fluorescence signal measuredafter the solid support capture of three different sizes of amplicon bythe indicated probe. The fluorescence signal is shown on the left of thepanel as a percentage of the signal measured in experiments using alinear (non-bridging) control probe for capture of these targets.

FIG. 43A shows a schematic diagram of a structure in the ampliconderived from HCV type 1a (residues 136 to 213 of SEQ ID NO:124) alignedwith bridging probe having two seven-nucleotide regions ofcomplementarity (SEQ ID NO:120). The regions that are complementary asaligned to the target are indicated by a black line between the strands.

FIG. 43B shows a schematic diagram of a structure in the ampliconderived from HCV type 1b (residues 22 to 125 of SEQ ID NO:125) alignedwith bridging probe having two 7 or 8 nucleotide regions ofcomplementarity (SEQ ID NOS:121 and 122, respectively). The regions thatare complementary as aligned to the target are indicated by a black linebetween the strands.

FIG. 44A shows a graph depicting the fluorescence signal measured afterthe solid support capture of the amplicons derived from HCV types 1a,1b, 2a/c and 3a by the indicated probe. The amplicons used in eachcapture test are indicated below each bar. The fluorescence signal isshown on the left of the panel as a percentage of the signal measured inexperiments using a linear (non-bridging) control probe for capture ofthis target, with 1 being 100 percent.

FIG. 44B shows a graph depicting the fluorescence signal measured afterthe solid support capture of the amplicons derived from HCV types 1a,1b, 2a/c and 3a by the probes indicated at the top of each panel. Theamplicons used in each capture test are indicated below each bar. Thefluorescence signal is shown on the left of the panel as a percentage ofthe signal measured in experiments using a linear (non-bridging) controlprobe for capture of this target, with 1 being 100 percent.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full length codingsequence or by any portion of the coding sequence so long as the desiredenzymatic activity is retained.

The term “wild-type” refers to a gene or gene product which has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product which displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally-occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

The term “LTR” as used herein refers to the long terminal repeat foundat each end of a provirus (i.e., the integrated form of a retrovirus).The LTR contains numerous regulatory signals including transcriptionalcontrol elements, polyadenylation signals and sequences needed forreplication and integration of the viral genome. The viral LTR isdivided into three regions called U3, R and U5.

The U3 region contains the enhancer and promoter elements. The U5 regioncontains the polyadenylation signals. The R (repeat) region separatesthe U3 and U5 regions and transcribed sequences of the R region appearat both the 5′ and 3′ ends of the viral RNA.

The term “oligonucleotide” as used herein is defined as a moleculecomprised of two or more deoxyribonucleotides or ribonucleotides,preferably more than three, and usually more than ten. The exact sizewill depend on many factors, which in turn depends on the ultimatefunction or use of the oligonucleotide. The oligonucleotide may begenerated in any manner, including chemical synthesis, DNA replication,reverse transcription, or a combination thereof.

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be the to have 5′ and 3′ ends.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points towards the 5′ end of the other,the former may be called the “upstream” oligonucleotide and the latterthe “downstream” oligonucleotide.

The term “primer” refers to an oligonucleotide which is capable ofacting as a point of initiation of synthesis when placed underconditions in which primer extension is initiated. An oligonucleotide“primer” may occur naturally, as in a purified restriction digest or maybe produced synthetically.

A primer is selected to have on its 3′ end a region that is“substantially” complementary to a strand of specific sequence of thetemplate. A primer must be sufficiently complementary to hybridize witha template strand for primer elongation to occur. A primer sequence neednot reflect the exact sequence of the template. For example, anon-complementary nucleotide fragment may be attached to the 5′ end ofthe primer, with the remainder of the primer sequence beingsubstantially complementary to the strand. Non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementarity with the sequence of thetemplate to hybridize and thereby form a template primer complex forsynthesis of the extension product of the primer.

As used herein, the terms “hybridize” and “hybridization” refer to theannealing of a complementary sequence to the target nucleic acid (thesequence to be detected) through base pairing interaction (Marmur andLane, Proc. Natl. Acad. Sci. USA 46:453 [1960] and Doty et al., Proc.Natl. Acad. Sci. USA 46:461 [1960]). The terms “annealed” and“hybridized” are used interchangeably throughout, and are intended toencompass any specific and reproducible interaction between anoligonucleotide and a target nucleic acid, including binding of regionshaving only partial complementarity and binding interactions that makeuse of non-canonical interactions for stability and/or specificity.

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” Certain bases not commonly found innatural nucleic acids may be included in the nucleic acids of thepresent invention and include, for example, inosine and 7-deazaguanine.Complementarity need not be perfect; stable duplexes may containmismatched base pairs or unmatched bases. Those skilled in the art ofnucleic acid technology can determine duplex stability empiricallyconsidering a number of variables including, for example, the length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

The term “non-canonical” as used in reference to nucleic acids indicatesinteractions other than standard, or “Watson-Crick” base pairing,including but not limited to G-T and G-U base pairs, Hoogsteininteractions, triplex structures, quadraplex aggregates, and multibasehydrogen bonding such as is observed within nucleic acid tertiarystructures, such as those found in tRNAs.

The stability of a nucleic acid duplex is measured by the meltingtemperature, or “T_(m).” The T_(m) of a particular nucleic acid duplexunder specified conditions is the temperature at which on average halfof the base pairs have disassociated.

The term “probe” as used herein refers to an oligonucleotide which formsa duplex structure or other complex with a sequence in another nucleicacid, due to complementarity or other means of reproducible attractiveinteraction, of at least one sequence in the probe with a sequence inthe other nucleic acid.

The terms “signal probe” and “signal oligonucleotide,” as used herein,are used interchangeably in reference to any oligonucleotide that isprovided to permit detection of the progress or products of a reactionor interaction. A signal probe may be labeled or unlabeled, and may bemodified or left unmodified by the mechanism of the reaction.

The term “label” as used herein refers to any atom or molecule which canbe used to provide a detectable (preferably quantifiable) signal, andwhich can be attached to a nucleic acid or protein. Labels may providesignals detectable by fluorescence, radioactivity, colorimetry,gravimetry, X-ray diffraction or absorption, magnetism, enzymaticactivity, and the like.

The terms “target nucleic acid” and nucleic acid substrate” are usedherein interchangeably and refer to a nucleic acid molecule which whendenatured and allowed to renature (i.e., to fold upon itself by theformation of intra-strand hydrogen bonds), forms at least one foldedstructure. The nucleic acid target may comprise single- ordouble-stranded DNA or RNA.

As used herein, the term “folded target” refers to a nucleic acid strandthat contains at least one region of secondary structure (i.e., at leastone double stranded region and at least one single-stranded regionwithin a single strand of the nucleic acid). A folded target maycomprise regions of tertiary structure in addition to regions ofsecondary structure.

The term “substantially single-stranded” when used in reference to anucleic acid target means that the target molecule exists primarily as asingle strand of nucleic acid in contrast to a double-stranded targetwhich exists as two strands of nucleic acid which are held together byinter-strand base pairing interactions.

Nucleic acids form secondary structures which depend on base-pairing forstability. When single strands of nucleic acids (single-stranded DNA,denatured double-stranded DNA or RNA) with different sequences, evenclosely related ones, are allowed to fold on themselves, they assumecharacteristic secondary structures. An alteration in the sequence ofthe target may cause the destruction of a duplex region(s), or anincrease in stability of a thereby altering the accessibility of someregions to hybridization of the probes oligonucleotides. While not beinglimited to any particular theory, it is thought that individualmolecules in the target population may each assume only one or a few ofthe structures (i.e., duplexed regions). but when the sample is analyzedas a whole, a composite pattern from the hybridization of the probes canbe created. Many of the structures that can alter the binding of theprobes are likely to be only a few base-pairs long and would appear tobe unstable. Some of these structures may be displaced by thehybridization of a probe in that region; others may by stabilized by thehybridization of a probe nearby, such that the probe/substrate duplexcan stack coaxially with the target intrastrand duplex, therebyincreasing the stability of both. The formation or disruption of thesestructures in response to small sequence changes results in changes inthe patterns of probe/target complex formation. Temperatures in therange of 20 to 55° C., with the range of 20 to 40° C. being particularlypreferred, are suitable temperatures for the practice of the method ofthe invention.

The term “sequence variation” as used herein refers to differences innucleic acid sequence between two nucleic acid templates. For example, awild-type structural gene and a mutant form of this wild-type structuralgene may vary in sequence by the presence of single base substitutionsand/or deletions or insertions of one or more nucleotides. These twoforms of the structural gene vary in sequence from one another. A secondmutant form of the structural gene may exist. This second mutant formvaries in sequence from both the wild-type gene and the first mutantform of the gene. It is noted, however, that the invention does notrequire that a comparison be made between one or more forms of a gene todetect sequence variations. Because the method of the inventiongenerates a characteristic and reproducible pattern of complex formationfor a given nucleic acid target, a characteristic “fingerprint” may beobtained from any nucleic target without reference to a wild-type orother control. The invention contemplates the use of the method for both“fingerprinting” nucleic acids without reference to a control andidentification of mutant forms of a target nucleic acid by comparison ofthe mutant form of the target with a wild-type or known mutant control.

The terms “structure probing signature,” “hybridization signature” and“hybridization profile” are used interchangeably herein to indicate themeasured level of complex formation between a folded target nucleic acidand a probe or set of probes, such measured levels being characteristicof the folded target nucleic acid when compared to levels of complexformation involving reference targets or probes.

The term “nucleotide analog” as used herein refers to modified ornon-naturally occurring nucleotides such as 7-deaza purines (i.e.,7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs include base analogsand comprise modified forms of deoxyribonucleotides as well asribonucleotides. As used herein the term “nucleotide analog” when usedin reference to targets present in a PCR mixture refers to the use ofnucleotides other than dATP, dGTP, dCTP and dTTP; thus, the use of dUTP(a naturally occurring dNTP) in a PCR would comprise the use of anucleotide analog in the PCR. A PCR product generated using dUTP,7-deaza-dATP, 7-deaza-dGTP or any other nucleotide analog in thereaction mixture is the to contain nucleotide analogs. “Oligonucleotideprimers matching or complementary to a gene sequence” refers tooligonucleotide primers capable of facilitating the template-dependentsynthesis of single or double-stranded nucleic acids. Oligonucleotideprimers matching or complementary to a gene sequence may be used inPCRS, RT-PCRs and the like. As noted above, an oligonucleotide primerneed not be perfectly complementary to a target or template sequence. Aprimer need only have a sufficient interaction with the template that itcan be extended by template-dependent synthesis.

The term “cleavage structure” as used herein, refers to a structurewhich is formed by the interaction of at least one probe oligonucleotideand a target nucleic acid to form at least one region of duplex, theresulting structure being cleavable by a cleavage means, including butnot limited to an enzyme. The cleavage structure is a substrate forspecific cleavage by the cleavage means, in contrast to a nucleic acidmolecule which is a substrate for non-specific cleavage by agents suchas phosphodiesterases which cleave nucleic acid molecules without regardto secondary structure (i.e., no formation of a duplexed structure isrequired).

The term “cleavage means” as used herein refers to any means which iscapable of cleaving a cleavage structure, including but not limited toenzymes. The cleavage means may include native DNAPs having 5′ nucleaseactivity (e.g., Taq DNA polymerase, E. coli DNA polymerase 1) and, morespecifically, modified DNAPs having 5′ nuclease but lacking syntheticactivity. The ability of 5′ nucleases to cleave naturally occurringstructures in nucleic acid templates (structure-specific cleavage) isuseful to detect internal sequence differences in nucleic acids withoutprior knowledge of the specific sequence of the nucleic acid. In thismanner, they are structure-specific enzymes. The cleavage means is notrestricted to enzymes having solely 5′ nuclease activity. The cleavagemeans may include nuclease activity provided from a variety of sourcesincluding the Cleavase® enzymes, the FEN-1 endonucleases (including RAD2and XPG proteins), Taq DNA polymerase and E. coli DNA polymerase I. Thecleavage means of the present invention cleave a nucleic acid moleculein response to the formation of cleavage structures; it is not necessarythat the cleavage means cleave the cleavage structure at any particularlocation within the cleavage structure.

The term “structure-specific nucleases” or “structure-specific enzymes”refers to enzymes that recognize specific secondary structures in anucleic molecule and cleave these structures without the regard to thespecific sequences making up the structure.

The term “thermostable” when used in reference to an enzyme, such as a5′ nuclease, indicates that the enzyme is functional or active (i.e.,can perform catalysis) at an elevated temperature, i.e., at about 55° C.or higher.

The term “cleavage products” as used herein, refers to productsgenerated by the reaction of a cleavage means with a cleavage structure(i.e., the treatment of a cleavage structure with a cleavage means).

The term “target nucleic acid” refers to a nucleic acid molecule whichcontains a sequence which has at least partial complementarity with atleast one probe oligonucleotide. The target nucleic acid may comprisesingle- or double-stranded DNA or RNA.

The term “probe oligonucleotide” refers to an oligonucleotide whichinteracts with a target nucleic acid to form a complex. The complex mayalso comprise a cleavage structure. The term “non-target cleavageproduct” refers to a product of a cleavage reaction that is not derivedfrom the target nucleic acid. In the methods of the present invention,cleavage of the cleavage structure may occur within the probeoligonucleotide. The fragments of the probe oligonucleotide generated bythis target nucleic acid-dependent cleavage are “non-target cleavageproducts.”

The term “invader oligonucleotide” refers to an oligonucleotide thathybridizes to a target nucleic acid such that its 3′ end positions thesite of structure-specific nuclease cleavage within an adjacentlyhybridized oligonucleotide probe. In one embodiment its 3′ end has atleast one nucleotide of sequence that is identical the firsttarget-complementary nucleotide of the adjacent probe; these nucleotideswill compete for hybridization to the same nucleotide in a complementarytarget nucleic acid. In another embodiment, the invader oligonucleotidehas a single 3′ mismatched nucleotide, and hybridizes to an adjacent,but not overlapping, site on the target nucleic acid.

The term “substantially single-stranded” when used in reference to anucleic acid substrate means that the substrate molecule existsprimarily as a single strand of nucleic acid in contrast to adouble-stranded substrate which exists as two strands of nucleic acidwhich are held together by inter-strand base pairing interactions.

A “consensus gene sequence” refers to a gene sequence which is derivedby comparison of two or more gene sequences and which describes thenucleotides most often present in a given segment of the genes; theconsensus sequence is the canonical sequence.

The term “polymorphic locus” is a locus present in a population whichshows variation between members of the population (i.e., the most commonallele has a frequency of less than 0.95). In contrast, a “monomorphiclocus” is a genetic locus at little or no variations seen betweenmembers of the population (generally taken to be a locus at which themost common allele exceeds a frequency of 0.95 in the gene pool of thepopulation).

The term “microorganism” as used herein means an organism too small tobe observed with the unaided eye and includes, but is not limited tobacteria, virus, protozoans, fungi, and ciliates.

The term “microbial gene sequences” refers to gene sequences derivedfrom a microorganism.

The term “bacteria” refers to any bacterial species includingeubacterial and archaebacterial species.

The term “virus” refers to obligate, ultramicroscopic, intracellularparasites incapable of autonomous replication (i.e., replicationrequires the use of the host cell's machinery).

The term “multi-drug resistant” or “multiple-drug resistant” refers to amicroorganism which is resistant to more than one of the antibiotics orantimicrobial agents used in the treatment of the microorganism.

The term “non-contiguous,” when used to describe regions within a targetnucleic acid to be analyzed, is intended to mean that the regions areseparated by intervening nucleic acid (or non-nucleic acid spacers). Itis not intended that the present invention be limited by the size of theintervening nucleic acid (or the size of non-nucleic acid spacers).However, in preferred embodiments, the intervening sequence is at leastfive nucleotides in length.

The term “non-contiguous,” when used to describe regions within anucleic acid probe, means sequences capable of hybridizing to thenon-contiguous regions of target nucleic acid. It is not intended thatthe present invention be limited to probes having intervening nucleicacid; that is to say, the non-contiguous regions of a probe are definedfunctionally, with reference to their binding to non-contiguous regionsin a target, the target having intervening nucleic acid separating thenon-contiguous regions. Nonetheless, the probes of the present inventionmay have (but need not have) intervening nucleic acid (or a non-nucleicacid spacer).

The terms “intervening nucleic acid,” “intervening portion,”“intervening region,” “intervening nucleic acid sequence,” and“intervening sequence,” refer to nucleic acid (single-stranded ordouble-stranded), that separates two or more regions (e.g.,non-contiguous regions) within a nucleic acid sequence. Where thepresent invention employs a probe having one or more interveningsequences, such intervening sequences are to be distinguished from meresingle base mismatched nucleic acid, such that intervening sequences onthe probe are at least two nucleic acids in length.

The term “bridging” when used in conjunction with a type of nucleic acid(e.g., oligonucleotide, probe, primer, etc.), refers to a nucleic acidthat is made to contact non-contiguous sites on a folded target nucleicacid. For example, a bridging probe and a bridging primer may refer tooligonucleotides that hybridize across a structure for detection, or forsubsequent primer extension, respectively, although “primer” and “probe”may also be used to indicate other types of interactions or reactions.

The term “non-bridging” when used in conjunction with a type of nucleicacid (e.g., oligonucleotide, probe, primer, etc.), refers to an nucleicacid that is not intended to hybridize across, a structure (i.e., itcontains a region substantially complementary its hybridization partnernucleic acid).

The term “reactant” refers to any agent that can act upon either thetarget or non-target nucleic acids to create a detectable alterationfrom the original nucleic acid chemical or nucleotide composition.

The terms “catalyzed reaction” or “catalytic reaction” refers to anyaction on a nucleic acid that is catalyzed or enacted by a reactantother than the nucleic acid.

The terms “modified probe” and “modified oligonucleotide” refer toprobes that have been altered from their original composition by theaction of a reactant. Such alterations include but are not limited tocleavage as by a nuclease, elongation as by a polymerase, or joining toanother entity, either through a covalent interaction, such as byligation to another nucleic acid, or by chemical cross-linking to anentity such as a protein, a nucleic acid, a detectable moiety, or asolid support.

DESCRIPTION OF THE INVENTION

The methods of the present invention use the combined effects ofmismatch and folded structure on hybridization to provide a tool for thedetection of mutations and other polymorphisms in nucleic acids (e.g.,DNA and RNA). The simultaneous probing of the primary (sequence),secondary (simple folded) and tertiary (interactions between secondaryfolds) structures of substrate molecules is referred herein simply as“structure probing.” Rather than destroying secondary structures by highstringency conditions and target fragmentation, the methods of thepresent invention use conditions in which the formation ofintramolecular structures is favored, i.e., unfragmented target strandsin conditions of low stringency. Thus, the present method of probing isdesigned to detect variations between nucleic acids at any of theselevels in a single assay.

At temperatures below the melting range of duplexed nucleic acid (i.e.,below the melting temperature of long [ie., >100 bp] nucleic acids; thisis generally taken to be temperatures below about 85° C. for a nucleicacid of average G-C content), single-stranded nucleic acids undergo acomplex process of intramolecular folding. The first rapid step of thisprocess involves formation of short-range, or local stem-loopsstructures. Later in the folding process, formation of tertiary orglobal structure occurs as a result of interactions between differentlocal domains (Zarrinkar and Williamson, Science 265:928 [1994]) andZarrinkar and Williamson, Nat. Struct. Biol., 3:432 [1996]). The effectsof secondary structure of the target on probe binding is well documentedfor DNA and RNA molecules (Gamper et al., supra; Fedorova et al., FEBSLett. 302:47 [1992]; Lima et al., Biochem., 31:12055 [1992]; Godard etal, Nucl. Acids Res., 22:4789 [1994]; Zarrinkar and Williamson, [1994],supra; Parkhurst and Parkhurst, Biochem., 34:285 [1995]; and Schwille etal., Biochem., 35:10182 [1996]). Target sequences that form stableduplexes within intramolecular secondary structures can have probebinding constants 10⁵-10⁶ times lower than sequences that exists as asingle strands (Lima et al., supra). The reduction of the hybridizationconstant for structured regions is primarily due to a lower associationrate constant rather than a higher dissociation rate constant (Lima etal., supra; Gamper et al., supra and Parkhurst and Parkhurst, supra),supporting the model that the structures in the target are blockingaccess of the probe to the complementary region within the targetmolecule.

Mutations in the target sequence change both local and globalconformations of the molecule. It has been shown that the conformationsassumed by single strands of nucleic acids can be probed using astructure-specific nuclease that cleaves in response to the structuresthat are formed in a number of test reaction conditions. (Brow et al.,supra). Such cleavage creates a collection of product fragments thatreflect those structures and which are characteristic of the particularstrands. The structures that give rise to cleavage patterns are verysensitive to the precise nucleotide sequence of the strand, such thateven single base differences in nucleic acids that are several hundrednucleotides long create sufficient changes in the folded conformationsto be detectable in the resulting cleavage pattern (Brow et al., supra),and the changes in electrophoretic mobility in SSCP. As a result ofthese changes, some regions that were previously base paired may becomeunpaired and vice versa. By measuring probe hybridization rates it ispossible to determine whether or not any region of a target moleculeforms intramolecular structure. The examples below describe the use ofmultiple oligonucleotides to characterize DNA fragments (i.e., forstructure probing). This approach is diagrammed schematically in FIG. 1.

In FIG. 1, three different, but related, target nucleic acids areanalyzed using the structure probing assay of the present invention.Allele/Type 1 represents the prototypical target sequence (e.g., a wildtype allele of gene X); Allele/Types 2 and 3 represent different allelesof the same target sequence (e.g., two different allelic variants ofgene X). The thick regions labeled 1-5 along the three target nucleicacids represent the regions along the target that are complementary toprobes 1-5. Allele/Type 2 contains a single-base variant (e.g., a pointmutation) relative to Allele/Type I (represented by the small opencircle between regions 3 and 4 of Allele/Type 2). This variant does notappear in a region where a probe binds to the Type 2 target; however,this variant alters the secondary structure of the Type 2 moleculerelative to that of the Type 1 molecule such that region 3 of the Type 2molecule is essentially unavailable for hybridization with probe 3.Allele/Type 3 also contains a single-base variant (e.g., a pointmutation) relative to Allele/Type 1 (represented by the small opencircle within region 3 of Allele/Type 3). The variant in this moleculeis located within a probe binding region and reduces the efficiency withwhich probe 3 binds to the Type 3 molecule. The target nucleic acids arerendered substantially single-stranded (i.e., they are denatured, e.g.,by heating) and then permitted to form secondary structures (e.g., bycooling) and then hybridized with probes 1-5. The probe/target complexesare captured onto a solid support and the amount of target that binds toeach of probes 1-5 is determined for each target to generate a probestructure signature (also referred to as a hybridization signature orprofile). The schematic shown in FIG. 1 is intended to illustrate thatthe signal variation may come from probe/target mismatch, or from theformation of local structures that block probe binding sites (i.e.,regions on the target which are at least partially complementary to theprobe). tertiary structure, involving interactions between sequences atsome distance (even several hundred nucleotides) may also block binding,i.e., mutations at one site may influence probe binding hundreds ofnucleotides away, as is seen with the katG targets employed in Example1.

In the Examples below, the oligonucleotide probes include a biotinmoiety so that the labeled target DNAs that have formed a hybridizationcomplex with the probes can be captured by exposure to a solid supportcoated with streptavidin. When used for immobilization in this way, theprobes are referred to herein as “capture probes.” The labels on the DNAcan then be detected, with the amount of captured DNA reflecting theefficiency of the probe/target hybridization, and thus the strength of aparticular binding interaction.

In the Examples below, the solid support employed is a well of a 96-wellmicrotiter plate. This format was chosen for convenience; the methods ofthe present invention are not limited to the use of microtiter plates orany particular support. The present invention contemplates the use ofmany types of solid supports, including but not limited to beads,particles, dipsticks, membranes and silicon or glass flat surfaces. Itis also contemplated that the binding of the probe/target complexes tosurfaces may be through interactions with the target nucleic acid (e.g.,the use of biotinylated target nucleic acids), while a detectable labelmay be included on the probes.

In the embodiments presented herein, the affinity of the target nucleicacid (e.g., a DNA fragment of interest) for different probes is assessedby performing separate hybridization and solid support capturedeterminations for each probe sequence. It is envisioned thatdifferently labeled probes, e.g., with different fluorescent dyes orother detectable moieties, may be used together in a single complexformation reaction. Use of an instrument that can detect several typesof signal, such as a fluorimeter with the capacity to excite and detectat a variety of wavelengths, allows the signal contribution from each ofthe bound probes to be assessed.

In some typing applications, variants may have any one of severalsequences (and therefore structures) and still be classed as the sametype (e.g., in HCV, there are numerous sequence variants that areclassed as type 1b). If it is not necessary to separately identify thesubtypes within a type, a mixture of probes may be provided such that atleast one type of probe will interact with each of the different knownvariants. If the target interacts appropriately (i.e., with the expectedaffinity) with any probe in the mixture it can be deduced to be of abroad type without concern about the identity of the particular subtypevariant. In this way, genetic materials known to vary in sequencewithout affecting function or type (as do many rapidly changingpathogens) may be analyzed in a single assay without the need for acomplex matrix of probes or for sequence determination.

In the following discussion, the oligonucleotide probes are discussed ascapture probes. The use of this term is for convenience only, to avoidrepetition of the enumeration of the possible configurations for thismethod, and it is intended that each of the embodiments described belowmay be used in combination with any of the probe/target configurations(e.g., labeled probes and captured target DNA and vice versa) describedabove.

The probes used in the methods of the present invention may be usedwithout any prior analysis of the structure assumed by a target nucleicacid. In designing such an assay, one designs probes that would span theentire length of the target sequence, (i.e., they would be complementaryto regions of the target that are substantially evenly spaced across theentire length of the target). Probes designed in this way may be phasedto a variety of densities. For example, the probes may each shift inhybridization site by one or a few nucleotides, to give a very highresolution fingerprint of the target, or they may be designed tohybridize to adjacent but not overlapping regions, to give thoroughcoverage at a slightly lower resolution. Alternatively, they may bespaced at much larger intervals for a lower resolution screen. Thechoice of spacing will be dependent on the needs of the assay. A higherdensity fingerprint will have a greater likelihood of identifying anypossible polymorphism, and may be more suitable for situations wherecertainty in identification of single base changes is required (e.g.,identification of mutations associated with cancers and other diseases).When genotyping is to be performed on targets in which more variation isexpected (e.g., rapidly changing viruses), a lower density array may besufficient for accurate identification. The examples below provide suchan analysis for the identification of Hepatitis C viral types. For anygiven case, it can be determined empirically using appropriatelyselected reference target molecule whether a chosen probe or array ofprobes can distinguish between genetic variants sufficiently for theneeds of a particular assay. Once a probe or array of probes isselected, the analysis of which probes bind to a target, and howefficiently these probes bind (i.e., how much of probe/target complexcan be detected) allows a hybridization signature of the conformation ofthe target to be created. One possible format for such a signature is asa graph of the measured amounts of a complex formed between the targetand each probe, as shown in FIGS. 4, 7, 8, and 9. It is not intendedthat the structure probing or hybridization signature be limited to theuse of the column graphs shown in these figures. It is contemplated thatthe signature may be stored, represented or analyzed by any of themethods commonly used for the presentation of mathematical and physicalinformation, including but not limited to line, pie, or area graphs or3-dimensional topographic representations. The data may also be used asa numerical matrix, or any other format that may be analyzed eithervisually, mathematically or by computer-assisted algorithms.

The resulting signatures of the nucleic acid structures serve assequence-specific identifiers of the particular molecule, withoutrequiring the determination of the actual nucleotide sequence. Whilespecific sequences may be identified by comparison of their signature toa reference signature, the use of algorithms to deduce the actualsequence of a molecule by sequence-specific hybridization (i.e., at highstringency to eliminate the influence of secondary and tertiarystructures) to a complete matrix (i.e., probes that shift by a singlenucleotide position at each location of an array), is not a feature orrequirement, or within the bounds of the methods of the presentinvention.

It is contemplated that information on the structures assumed by atarget nucleic acid may be used in the design of the probes, such thatregions that are known or suspected to be involved in folding may bechosen as hybridization sites. Such an approach will reduce the numberof probes that are likely to be needed to distinguish between targets ofinterest.

There are many methods used to obtain structural information involvingnucleic acids, including the use of chemicals that are sensitive to thenucleic acid structure, such as phenanthroline/copper, EDTA-Fe²+.cisplatin, ethylnitrosourea, dimethyl pyrocarbonate, hydrazine, dimethylsulfate, and bisulfite. Such chemical reagents may cause cleavage basedon structure, or they may cause nucleotide modification that cansubsequently be detected, such as by pausing or blocking of reversetranscriptase or other DNA polymerase copying, or by fingerprinting orother chromatography methods. Those skilled in the art are familiar withnumerous additional methods for the detection of nucleotidemodifications within a nucleic acid strand.

Enzymatic probing can be done using structure-specific nucleases from avariety of sources. Duplex-specific nucleases such as cobra venom V,nuclease have been widely used in the analysis of RNA structures (Seee.g, Lowman and Draper, J. Biol. Chem., 261:5396 [1986]). In addition,suitable 5′ nucleases include the Cleavase® enzymes (Third WaveTechnologies, Inc., Madison, Wis.), Taq DNA polymerase, E. coli DNApolymerase I, and eukaryotic structure-specific endonucleases (e.g.,human, murine and Xenopus XPG enzymes, yeast RAD2 enzymes), murine FEN-1endonucleases (Harrington and Lieber, Genes and Develop., 3:1344 [1994])and calf thymus 5′ to 3′ exonuclease (Murante et al., J. Biol. Chem.,269:1191 [1994]). In addition, enzymes having 3′ nuclease activity suchas members of the family of DNA repair endonucleases (e.g., the RrpIenzyme from Drosophila melanogaster, the yeast RAD1/RAD10 complex and E.coli Exo III), are also suitable for examining the structures of nucleicacids. In Example 3, the use of the CFLP® method for identifying regionsof folding in PCR amplified segments of the HCV genome is described.

If analysis of structure as a step in probe selection is to be used fora segment of nucleic acid for which no information is availableconcerning regions likely to form secondary structures, the sites ofstructure-induced modification or cleavage must be identified. It ismost convenient if the modification or cleavage can be done underpartially reactive conditions (i.e., such that in the population ofmolecules in a test sample, each individual will receive only one or afew cuts or modifications). When the sample is analyzed as a whole, eachreactive site should be represented, and all the sites may be thusidentified. Using a CFLP® cleavage reaction as an example, when thepartial cleavage products of an end labeled nucleic acid fragment areresolved by size (e.g., by electrophoresis), the result is a ladder ofbands indicating the site of each cleavage, measured from the labeledend. Similar analysis can be done for chemical modifications that blockDNA synthesis; extension of a primer on molecules that have beenpartially modified will yield a nested set of termination products.Determining the sites of cleavage/modification may be done with somedegree of accuracy by comparing the products to size markers (e.g.,commercially available fragments of DNA for size comparison) but a moreaccurate measure is to create a DNA sequencing ladder for the samesegment of nucleic acid to resolve alongside the test sample. Thisallows rapid identification of the precise site of cleavage ormodification.

Two approaches have commonly been applied to elucidate nucleic acidsecondary structures: physical approaches, such as analysis of crystalstructure or NMR, and analytical approaches, such as comparative orphylogenetic analysis. Physical analysis remains the only way to get acomplete determination of a folded structure for any given nucleic acid.However, that level of analysis is impractical if the goal is to analyzea large number of molecules. By far, the most often used method ofanalyzing biological nucleic acids is a phylogenetic, or comparativeapproach. This method of analysis is based on the biological paradigmthat functionally homologous sequences will adopt similar structures.Sequences are screened for sequence conservation, stem-loopconservation, and for compensatory sequence changes that preservepredicted structures. Unfortunately, such analysis can only be appliedwhen the number of related sequences is large enough for statisticalanalysis.

The efficient analysis of single nucleic acids requires the use ofmultiple tools. Many of the available tools can give partial informationon the possible structures assumed by a given molecule. As stated above,these methods include enzymatic analysis, chemical structure probing,and computer based analysis of regions of base pairing. In addition,deletion studies, in which portions of a linear molecule are deleted andthe effects on the folding are analyzed by the above-cited methods, canhelp identify with more certainty those regions of a nucleic acid thatinteract with each other. None of these methods in isolation can providesufficient physical information to identify with certainty anynon-contiguous regions that will be in close enough proximity to besimultaneously contacted by a bridging oligonucleotide. For example, oneof the most commonly used nucleic acid folding programs, “mfold” (Zukcr,Science 244:48 [1989]; Jaeger et al., Proc. Natl. Acad. Sci. USA,86:7706 [1989]; Jaeger et al., Meth. Enzymol. 183:281[1990]) usespreviously determined physical measurements for the effects of varioussecondary structure features, such as basepair combinations, loops,bulges, etc., on the stability of folded structures to predictstructures that have the lowest possible free energy. This approach isreferred to as an energy minimization approach (See, Gaspin and Westhof,J. Mol. Biol. 254:163 [1995] for review). While mfold and othercomputer-based folding algorithms can be made to present only thosestructures that are most likely to form (e.g., that arethermodynamically favored), when the software is permitted to showstructures that are even slightly less energetically favorable, thereare usually dozens of such structures predicted for any given nucleicacid strand. Even though these structures may be very stable, and may infact be proven to exist in nature, they are referred to as “suboptimal”structures, because they are calculated to have a less favorable freeenergy based on the software parameters. Using information derived fromthe other methods (e.g., analyzing folded structures or by physicalmethods), allows the number of structures to be pared down dramatically,from many, many possible structures, to a few probable ones.

One additional software-based approach involves tallying the number ofpairing partners available for each base within a collection ofsuboptimal structures predicted for a given nucleic acid strand (Zukerand Jacobson. Nucl. Acids Res. 23:2791 [1995]). The pairing number, or“p-num” for each base gives a quantitative measure of the fidelity ofpairing, i.e., the number of possible pairing partners, of each baseposition. It has been observed that predicted structures containingbases with p-nums that are lower than those of surrounding regions havea stronger correlation with structures that have been verified byphysical or phylogenetic conservation data. Therefore using mfold andp-num together can help simplify the task of identifying structures thatmay be assumed by a nucleic acid strand. Both p-num and mfold areavailable commercially (Genetics Computer Group, Madison, Wis.).

A significant limitation of the energy minimization programs for nucleicacids folding is that all of them, including mfold, use greatlysimplified thermodynamic models that include energy parameters that arenot well defined. The result is that the predicted optimal structuresmay not correspond to the actual conformation of the nucleic acid insolution. A partial solution to this is to extend the number of computedstructures to include those that have suboptimal energies, therebyincreasing the chances that one of them has better correlation with areal one. This step may produce an large number of possible structures,and identification of actual structures may be difficult without otheranalytical tools. For example, the mfold predictions done for the HCVtype 1a amplicon, as described in Example 8, resulted in 32 predictedstructures.

Efficient screening of the suboptimal structures can be accomplished byincorporating constraints derived from experimental data or phylogeneticanalysis into the computer algorithm. The use of structure specificnucleases having well characterized specificity have an advantage thatthe site of cleavage can convey additional information based on thestructural requirements for cleavage. This is illustrated here bydiscussion of information potentially gained by cleavage with a 5′nuclease, Cleavase® I nuclease, but the same deductive approach isequally applicable and useful for other structure-specific cleavageagents for which a substrate structure is well defined (i.e., it isknown where in the structure the cleavage can occur). The specificity ofCleavase® enzymes is such that cleavage occurs at the 5′ ends of hairpinduplexes, after the first base pair (Lyamichev et al., supra). Thismeans that any cleavage site identifies both a base that must be pairedin the structure, and that the base to which it pairs must be downstreamin the strand. This can expressed as follows: if there is a cleavagesite at position i, then nucleotide i is base paired with nucleotide jwhere j>i. Entering into mfold the parameters ‘f i 0 2’ and ‘p i−i+11−i−1’ specifies that nucleotides i and i+1 should be basepaired tosomething (not to each other) and that i and i+1 can not be basepairedwith nucleotides from 1 to i−1, respectively. This type of parameter canbe considered a “soft” parameter because, while base pairing isrequired, the specific pairing partners of i and i+1 are left undefined,thereby allowing the suboptimal foldings generated using theseparameters to predict multiple basepairing partners of thesenucleotides. This allows the use of existing constraint parameterswithout modification of the folding algorithm to predict only thosestructures that correlate with the cleavage data. If cleavage occurs atposition i, then a series of structures can be calculated to explain itusing the following constraints, ‘f i 0 1’ (nucleotide i is forced to bebase paired) and ‘p 1 0 i-1’ (prohibiting nucleotides from 1 to i−1 tobe base paired). For example, to generate structures that could beresponsible for a major cleavage site at position 90 of HCV1a DNA,folding of 244 nt DNA fragment of HCV1a (FIG. 15) (SEQ ID NO:124) wasdone using mfold version 2.3 (http://www.ibc.wustl.edu/˜zuker) withconstraints ‘f 90 0 1’ and ‘p 1 0 89’ predicting structure shown in FIG.16A (SEQ ID NO:128). It is important that this structure not onlypredicts a cleavage site at position 90, but also explains cleavages atpositions 102-103, 161 and 173, making it a good candidate to representactual base pairing in the DNA molecule. The structure shown in FIG. 16A(SEQ ID NO:128) does not explain cleavage sites at positions 118-119 and173. To reveal corresponding structures, the folding was done usingconstraints ‘f 118 0 1’ and ‘p 1 0 117’ (nucleotides 1-117 are not basepaired and nucleotide 118 is base paired) with one of resultingstructures shown in FIG. 16B (SEQ ID NO:128). Again this structure notonly reasonably predicts cleavage site at position 117-118 but alsoshows how cleavage at position 123 may happen. The same two structureswere identified in the development of the experiments described inExample 8, using manual comparison of the cleavage sites and the 32suboptimal folds. By either method, the knowledge of the structurespecificity of the 5′ nuclease made it possible to eliminate fromconsideration, all predicted structures that would require the cleavagesites to vary from the known substrate structure. This reduced the fieldof possible structures from 32 to 2. Use of additional enzymes, such as3′ nucleases, or duplex specific chemical agents, that can identifyother positions that must be base-paired within a structure can furthernarrow the field.

Among different baseparing partners predicted for nucleotide i, the onethat is responsible for the Cleavase® site at position i can bedetermined experimentally by using a combined deletion/mutationtechnique referred to as “PCR walking.”The PCR walking technique isbased on CFLP analysis of PCR subfragments that are shorter variants ofthe analyzed sequence, variants that include only nucleotides from 1 tothe selected partner of nucleotide i. For example, if the softconstraints cause mfold to predict that nucleotide 25 is paired withnucleotide 67, the PCR walk subfragments would include nucleotides 1-67.For each tested basepair, two subfragment variants are generated; onehaving a wild type sequence and another having the putative basepairingpartner for nucleotide i (i.e., the 3′ terminal nucleotide) substitutedwith a base that is not complementary to i. In the example above, thebase to be substituted would be at position 67.

CFLP® cleavage analysis is then performed on both of these subfragments.If the putative pairing partner does in fact basepair to i, then thewild type PCR subfragment would show cleavage immediately after i, butthe substituted variant would show either a loss of cleavage, or ashifting of the cleavage site. If cleavage is the same in bothsubfragments, then i is pairing elsewhere; if cleavage at the originalsite is absent in both fragments, then the original pairing partner waslikely to have been in the region deleted to make the subfragments. Oncebasepairing partner j of nucleotide i is determined, this informationcan be used as a “hard” constraint in the mfold program, forcingnucleotides 1 and i+1 be basepaired with nucleotides j and j−1.

Similar procedure can be repeated for each cleavage site, therebygenerating a set of CFLP®-defined constraints. Compatible constraintscan be combined into groups so that each group would define analternative structure of the molecule.

This procedure was used to find alternative secondary structures of 244nucleotide RT-PCR fragment of HCV 1b 5′UTR region. Energy minimizationfolding of HCV 1b fragment using the mfold program without constraintsgenerated 29 structures, with difference in free energy between the twomost stable structures of only 1.3%. Folding with soft constraints ‘f 900 2’ and ‘p 90-91 1-89’, dictated by the major cleavage site at position90, produced 28 structures (the difference between two most stablestructures being 1.4%), 17 of which predicted baseparing betweennucleotides 90 and 135, 4 of which predicted basepairing betweennucleotides 90 and 105, another 4 predicted a 90-184 basepair, 2predicted a 90-229 basepair, and 1 predicted a 90-198 basepair. PCRwalking analysis showed that cleavage at position 90 can be explained bybasepairing between nucleotides 90 and 135. Using this information as a“hard” constraint ‘f 90 135 2’ forces basepairing between nucleotides90-91 and 134-135. Folding with this constraint resulted in 18structures with difference in ΔG between optimal and suboptimalstructures still only 1.4%.

A similar study for a cleavage site at position 161 showed it to pairwith nucleotide 205. The constraints for cleavage sites 90 and 161 arecompatible, meaning that they do not result in mutually exclusivestructures, and can be combined together. Running the folding programwith both constraints, generated 13 structures and increased thediscrimination between the two most stable structures to 3.4%. Thisprocess was continued by adding two new constraints for cleavage sitesat positions 33 and 173, decreasing the number of predicted structuresto 10, and increasing the difference in free energy between the optimaland first suboptimal structures to 7.2%, increasing the certainly thatthe optimal structure is likely to be form by the molecule.

In summary, we describe here a stepwise process for the analysis ofnucleic acid structure without the use of the expensive and timeconsuming traditional techniques such as crystallography and nuclearmagnetic resonance. This process comprises the steps of: a) performingCFLP® analysis to identify nucleotides that are basepaired on the 5′sides of stems; b) using this partial basepair information as a “softconstraint” in a fold-prediction program such as mfold to produceschematic diagrams (or other suitable output) of possible foldedconformations that are consistent with the CFLP® data; c) using PCRdeletion and directed mutagenesis to confirm the identities of thenucleotides on the 3′ sides of stems, to which the 5′ side nucleotidesare hydrogen bonded; and d) using this full basepair information as a“hard constraint” in the fold-prediction program to produce a highlyrefined set of predicted structures. Depending on the complexity of thedata generated at each step, one or more of steps (a) through (d) may beomitted in any particular application. As noted above, a number ofphysical analytical methods may be combined with a number of secondarystructure prediction algorithms to perform this type of analysis; theuse of CFLP® cleavage method in conjunction with the mfold software isdiscussed here as a convenient example and is not presented as alimitation on the scope of the present invention. The structureinformation gained in this process may be used not only is design of thestructure probes of the present invention, but also in the improvementof CFLP®, SSCP, and like mutation detection methods, and in theimprovement of many hybridization-based methods that suffer as aconsequence of target strand-structure interference, including but notlimited to the polymerase chain reaction, dideoxynucleotide-chaintermination sequencing, sequencing by hybridization, and other chiphybridization methods, ribozyme nucleic acid cleavage, and antisensemanipulation of gene expression in vivo.

In addition to the structural mapping methods described above, there areseveral methods based on the actions of polymerizing enzymes that may beused to gain structural information. It has long been observed thatreverse transcriptases can have difficulty polymerizing through RNAsecondary structures. For this reason, reverse transcriptases that canbe used at high temperatures have been sought (Myers et al., Biochem.,30:7661 [1991]), in order to facilitate full-length reversetranscription before cloning or PCR amplification. By intentionallyusing polymerases that produce such pausing effects, structures formedin a template strand may be mapped by the location of the pause sites(e.g., by extension of a labeled primer).

Another approach based on the use of DNA polymerases takes advantage ofthe observation that some DNA polymerases, upon encountering a fold inthe template strand, will apparently polymerize across a structure by amechanism that has been termed “strand switching,” thereby deleting thecomplement of the structured intermediate sequence. Though anunderstanding of the mechanism of strand switching is not necessary inorder to practice the present invention, it is believed that strandswitching involves some degree of displacement synthesis, such that asmall portion of a sequence (even to the level of one base), isduplicated, followed by a branch migration that pairs the 3′ end of theelongated strand with sequences on the far side of the templatestructure (Patel et al., Proc. Natl. Acad. Sci. USA 93:2969 [1996]).This mechanism can conceivably be used for structure mapping in at leasttwo ways. For example, if the 3′ side of a structure has been mappedusing a 3′ nuclease in a CFLP® reaction, as discussed above, a primermay be designed such that the 3′ end of the primer is poised topolymerize either along or across the structure-forming region. Inaddition to its template complementary sequence, the primer may besupplied with one or a few degenerate nucleotides (e.g., two or morenucleotides at the same position on different copies of the primer) onthe 3′ end, to provide opportunity for strand switching, regardless ofthe downstream sequence. The primer may then be extended underconditions favoring strand switching (Patel et al., supra). Theisolation (e.g., by cloning and sequencing) of such sites shouldidentify the sequences that are coming together to form the foldedstructures, thus facilitating bridge oligonucleotide design. A secondapproach is similar, but without the use of primers adjacent to anyparticular putative structure. In this embodiment, a strand to beanalyzed is primed using a normal primer, and synthesis is carried outin the same or similar strand switch favoring conditions. The use ofconditions that favor base misincorporation (e.g., by the use ofmanganese in the synthesis reactions), and therefore promote pausing ofthe polymerase, would provide additional opportunity for branchmigration and strand switching. The analysis of the junction sites wouldthen follow as with the first approach. By these methods, both sides ofa cleavage structure could be identified. It is also expected thatalternative pairing partners for various sequences would be representedin the collection of molecules created.

To distinguish between related nucleic acids, the regions that showdifferent sites of cleavage or modification have the highest probabilityof having secondary structures that will respond differently to probesin the methods of the present invention. This is for two reasons. First,the cleavage or modification is physical evidence that a structure mayform at a given site under the conditions of the cleavage ormodification assay. Second, the structures that are detected by theCFLP® method have been found to be predominantly local (i.e., formedfrom sequences that are close to each other along the nucleic acidstrand, Brow et al., supra), so that changes observed are likely to becaused by base changes near the altered cleavage site. By designingoligonucleotide probes to hybridize or complex with the regions showingdifferent sites of cleavage or modification there is a higherprobability of finding either a base change (primary structurevariation) or a folding change (secondary structure variation) that willaffect the complexing of the probe to that site, thus facilitating thedistinction between the comparison targets. Because of the complexnature of the folded structure formation as described above and becauseany given probe may interact with the target in a number of ways,choosing a probe in this way is not a guarantee that any particularprobe will provide a diagnostic distinction. This is offered as a guideto increase the probability that it will. When working with anuncharacterized target or set of targets, the use of a multiplicity ofsuch probes will give the most distinctive signature of probe/targetcomplex formation.

In one embodiment, it is preferred that the probes used in the methodsof the present invention be short enough to provide distinctivehybridization signatures for variants of a target. Probes longer thanabout 20 nt (e.g., 20 to 40 nt) can interact with target nucleic acidsin a specific manner at elevated temperatures (e.g., higher than about40° C.) and thus are suitable for use in the present methods. However,probes in this size range may interact with multiple sites on the targetif the reaction is performed below about 40° C., reducing thedistinction between variants. If this is the case, higher reactiontemperatures or more stringent solution conditions (e.g., lower salt,the inclusion of helix-destabilizing agents such as dimethyl sulfoxideor formamide) may prove useful in enhancing the distinction betweentargets. In a particularly preferred embodiment, the method of thepresent invention is performed at ambient temperatures (e.g., 20 to 25°C.). When the assay is performed at room temperature, small probes withT_(m)s of 40° C. or less (e.g., 10 to 20 nt) can provide thediscrimination necessary, as shown in the examples below. Probes in thissize range are also less likely to fold on themselves under the reactionconditions, an effect that would reduce the binding efficacy of a probewithout regard to the structure of the target.

As stated above, the capture probe may interact with the target in anynumber of ways. For example, in another embodiment, the capture probesmay contact more than one region of the target nucleic acid. When thetarget nucleic acid is folded as described, two or more of the regionsthat remain single stranded may be sufficiently proximal to allowcontact with a single capture probe. The capture oligonucleotide in sucha configuration is referred to herein as a “bridge” or “bridging”oligonucleotide, to reflect the fact that it may interact with distalregions within the target nucleic acid. The use of the terms “bridge”and “bridging” is not intended to limit these distal interactions to anyparticular type of interaction. It is contemplated that theseinteractions may include non-canonical nucleic acid interactions knownin the art, such as G-T base pairs, Hoogstein interactions, triplexstructures, quadraplex aggregates, and the multibase hydrogen bondingsuch as is observed within nucleic acid tertiary structures, such asthose found in tRNAs. The terms are also not intended to indicate anyparticular spatial orientation of the regions of interaction on thetarget strand, i.e., it is not intended that the order of the contactregions in a bridge oligonucleotide be required to be in the samesequential order as the corresponding contact regions in the targetstrand. The order may be inverted or otherwise shuffled.

It is known that synthetic oligonucleotides can be hybridized tonon-contiguous sequences in both RNA and DNA strands, in a manner thateither causes the intervening sequence to loop out, or that bridges thebase of an internal folded structure (Richardson et al., J. Am. Chem.Soc., 113:5109 [1991]; Francois et al., Nucl. Acid. Res., 22: 3943[1994]). However, these references do not suggest the design or use ofbridging oligonucleotides that can distinguish between the differentfolded structures, or that bind with significantly reduced efficiencywhen the intervening sequence is unstructured. The present inventionprovides methods for the use and design of bridge capture probes withminimally stable regions of complementarity to make these bridge probessensitive to changes in the target strand structure. Minimal stability(i.e., with a very low melting temperature), may be created in a numberof ways, including by the use of short lengths of complementarity, lowG-C basepair content, and/or the use of base analogs or mismatches toreduce the melting temperature. To test the effects of variations in thetarget structure on the efficiency of capture with different lengths ofbridge probes, three test molecules were created; these are shown inschematic representation in FIG. 10. Test molecule #80 (SEQ ID NO:39)has a long segment of self complementarity and when folded as shown, the8 basepair hairpin formed by this oligonucleotide is further stabilizedby a “tri-loop” sequence in the loop end (i.e., three nucleotides formthe loop portion of the hairpin) (Hiraro et al., Nucleic Acids Res.22(4):576 [1994]). In test molecule #81 (SEQ ID NO:40), the stem isinterrupted by 2 mismatches to form a less stable structure, and theregion of self-complementarity is entirely removed in test molecule #82(SEQ ID NO:41). All three of these molecules have identical targetregions for the binding of the capture oligonucleotides, and anexamination of their use is described in Example 6.

When a bridging oligonucleotide contacts sequences on either side of abasepaired stem, the structure formed is termed a three-way or three-armjunction. Such junctions have been studied extensively to determinetheir physical structure and to assess the differences that occur in thephysical structure when additional nucleotides are included in thesestructures. When extra nucleotides are included at the junction site,where the three strands come together (i.e., when a ‘bulged’ structureis formed), it has been shown that the structure is more flexible andthat some degree of coaxial stacking between the arms stabilized thestructure compared to the unbulged structure (See e.g., Zhong et al.,Biochem., 32:6898 [1993]; and Yang et al., Biochem., 35:7959 [1996]).The inclusion of two thymidine nucleotides in the portion of the probethat forms the junction is particularly preferred.

There are a number of approaches that may be used in the design orselection of bridging capture probes. As noted above, the term “captureprobes” is not intended to limit the application of the bridging probesof the present invention to the capture of a target strand onto a solidsupport. Additional applications of the bridging probes are described inthe Experimental Examples, below. Furthermore, for simplicity ofdiscussion and to avoid repetition, this section describes oneembodiment of the present invention, namely a process for creatingbridge oligonucleotides that interact with only two regions of a targetnucleic acid. It is not intended, however, that the invention be limitedto the use of oligonucleotides that have only two sites of interaction.It is contemplated that bridge oligonucleotides may be created that caninteract with many sites on a folded target molecule.

Bridge oligonucleotides may be created by the joining two or more shortoligonucleotide sequences. The creation of bridge oligonucleotides maybe based upon observations that these sequences have been determined tointeract with a given folded target when used in isolation, withoutlimitation to any particular nature of interaction, or they may bededuced to be capable of such interaction by virtue of sequencecomposition, complementarity, or like analysis. For convenience, suchsequences are termed herein “contact sequences,” to reflect the putativeability of such a sequence to contact the target molecule. Thedesignation of a particular sequence as a contact sequence is notintended to imply that the sequence is in contact, or is required tocontact a target in any particular embodiment.

In alternative embodiments, contact sequences may be joined bysynthesizing or otherwise creating a new oligonucleotide thatincorporates both sequences into a single molecule. In one embodiment,the sequences are joined contiguously within the bridge oligonucleotide(i.e., without any intervening nucleotides or other space-fillingmaterial). In another embodiment, the contact sequences arenon-contiguous, with the spacing provided by additional nucleotides. Ina preferred embodiment, the contact sequences are bridged by twothymidine nucleotides, as depicted in several of the bridging probes inFIG. 11A (SEQ ID NOs: 50, 39, 42-44, and 47-49, respectively). Inanother preferred embodiment, the contact sequences in the bridgingoligonucleotide are connected by a segment of nucleic acid containing aregion of self-complementarity, such that the bridging oligonucleotideitself contains a folded structure. A stem-loop folded structure withinthe bridge oligonucleotide, if situated opposite a stem in the targetnucleic acid, would permit the formation of a four-way Hollidaystructure, which is stabilized by coaxial stacking of the arms (Duckettet al., Cell 55:79 [1988]).

Alternatively, the bridge oligonucleotide may be created by linking theindividual sequences with non-nucleotide spacers such as those commonlyknown in the art, such as d-spacers (Glen Research Corp. (Sterling,Va.), or other chemical chains, such as polyethers (Cload and Shephartz,J. Am. Chem. Soc., 113:6324[1991]).

Contact sequences may also be linked to form the bridge probes postsynthetically, by enzymatic (e.g., ligation) or by chemical interactionto produce either covalent (e.g., cross-linked) or non-covalent bonds(e.g., affinity bonds such as formed in an antigen-antibodyinteraction).

The formation of the complexes between the probes and the targets may beperformed using a wide variety of solution conditions. Conditionsconsidered to be “low stringency” have been well defined in the areas ofhybridization to filters and membranes (Sambrook et al, MolecularCloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. [1989]) and to other solid supports,such as silicon or glass wafers, chips or slides (Maskos and Southern,Nucl. Acids Res., 20:1675 [19921]). It is contemplated that theformation of the complexes may be done in solution, before the bindingof either the target or the probe to a solid support, or it may be doneafter one of the molecules has been bound to the support. It isrecognized, and considered to be within the scope of the invention, thatthe kinetics and mechanics of complex formation may differ depending onwhether complex formation is performed in solution or on a solidsupport. The identity of the support would also be expected to influencethe complex formation. However, as long as complexes can be made to format detectable levels, a set of conditions is considered appropriate foruse in the present methods.

It is further contemplated that the complexes may be formed on nucleicacids that have not been isolated from a sample source, such as in livecells (in vivo) or in tissue samples (in situ). It is also contemplatedthat a nucleic acid found within a cell may be native to that cell, ormay be transferred into the cell (e.g., by viral infection, bylaboratory-induced transfection, or by in vivo transcription from anintroduced nucleic acid). The methods of the present invention asapplied to nucleic acids within cells are not limited to nucleic acidsof any particular origin or cell type.

A number of solid supports known in the art are contemplated for usewith the methods of the present invention. In the examples below, a96-well microtiter plate is used as a support medium. The method mayalso be applied to other supports nucleic acid commonly used for nucleicacid analyses, including but not limited to beads, particles, membranes,filters, dipsticks, slides, plates and microchips. Such supports may becomposed of a number of materials known to be compatible with nucleicacids analyses, including but not limited to agarose, styrene, nylon,glass and silicon.

Individual complex formation (i.e., assessing a single target with asingle probe) may be sufficiently informative for some applications. Inother applications, it may be desirable to use a number of probesagainst a single target. For a large number of probes, it may be usefulto use an array format, in which a large number of probes are bound to asurface in an ordered pattern. Means for creating such arrays onsurfaces such as glass slides and microchips are known in the art(Southern et al., Genomics 13:1008 [1992]; Chee et al., Science 274:610[1996]; and Foder et al., Science 251:767 [1991]; and U.S. Pat. Nos.5,436,327 to Southern et al., 5,429,807 to Matson et al. and 5,599,695to Pease et al., all of which are herein incorporated by reference).

A. Use of Bridging Oligonucleotides in Catalyzed Reactions

As discussed above, it is contemplated that any catalyzed reaction thatis specifically operative on a duplex formed between a target nucleicacid and a substantially complementary probe may be configured toperform on the bridging probe/folded target complex. Examplesdemonstrating the use of bridging probes in primer extension, ligationand structure-specific nuclease cleavage are provided below. Primerextension reactions and ligation reactions are well known in the art andthe basic method for performing these reaction are published (See e.g.,Sambrook et al, supra), as well as often being provided by themanufactures of the enzymes. The Invader™ invasive cleavage reaction isbased on the use of a structure-specific nuclease that is used to cleaveoligonucleotide probes once they hybridize to a target nucleic acid. Thenature of the reaction allows the cleavage of many copies of the probeoligonucleotide for each copy of the target nucleic acid. Completedescriptions of the technology and its variables are included in PCTInternational Application No. PCT/US97/01072 (WO 97/27214) andco-pending application Ser. Nos. 08/599,491, 08/682,853, 08/756,386,08/759,038, and 08/823,516, all of which are herein incorporated byreference. Briefly, The Invader™ assay is a method for detecting aspecific target sequence within a nucleic acid mixture. The assaydepends on the coordinate actions of at least two syntheticoligonucleotides, together constituting a probe system, and astructure-specific nuclease. The oligonucleotides of the probe systemmay be referred to as the signal oligonucleotide and the Invadermoligonucleotide. By the extent of their substantial complementarity tothe target strand, each of these oligonucleotides defines a specificregion of the target strand. These regions must be oriented such thatwhen the probe system is hybridized to the target strand, the Invader™oligonucleotide is upstream of the signal oligonucleotide and such thatthe Invader™ oligonucleotide sequence either overlaps with the probeoligonucleotide sequence by at least one nucleotide (i.e., the tworegions of the target nucleic acid defined by the oligonucleotides ofthe probe system share at least one nucleotide), or, when there is nooverlap, the two target regions defined by the oligonucleotides mustabut, and the 340 terminus of the Invader™ oligonucleotide must have asingle additional nucleotide that is not complementary to the targetstrand at that site.

The nuclease recognizes the structure formed by hybridization of theprobe system to the specific target nucleic acid and cleaves the signaloligonucleotide, the precise site of cleavage being dependent on theamount of its overlap with the Invader™ oligonucleotide. If the reactionis run such that the structure can partially disassemble to allowcleaved signal oligonucleotide to be replaced by intact signaloligonucleotide (e.g., performed at an elevated temperature to promoterapid dissociation and association of signal probes), then multipleprobes may be cleaved for each copy of the target nucleic acid, theamount of target present then being calculable from the rate of productaccumulation and the time of incubation.

The nucleases of the Invader™ assay include any nuclease capable ofspecifically recognizing the structure defined above, and cleavingwithin the signal oligonucleotide, thereby creating cleavage products.Such nucleases include, but are not limited to the 5′ nucleasesassociated with eubacterial DNA polymerases, and the DNArepair-associated nucleases of the FEN1, RAD2 and XPG classes.

The oligonucleotides of the Invaderm probe system may comprise DNA, RNA,PNA and combinations thereof, as well as modified nucleotides, universalbases, adducts, etc. They may be either fully or partially complementaryto their cognate target sequences. In addition, they may be labeled orunlabeled.

Detection may be by analysis of cleavage products or by analysis ofremaining uncleaved signal probe. Detection of the cleavage products maybe through release of a label. Such labels comprise: dyes; radiolabelssuch as ³²P; binding moieties such as biotin; haptens such asdigoxgenin; luminogenic, phosphorescent or fluorogenic moieties;fluorescent dyes alone or in combination with moieties that can suppressor shift emission spectra by fluorescence resonance energy transfer(FRET).

Cleavage products may be analyzed by physical separation (e.g., byelectrophoresis, hybridization or by selective binding to a support) orwithout physical separation (e.g., by changes in fluorescence inFRET-based analysis, or by change in rotation rate in solution influorescence polarization analysis).

Cleavage products can be used subsequently in any reaction or read-outmethod that can make use of oligonucleotides. Such reactions includeenzyme dependent modification reaction, such as ligation, tailing with atemplate-independent nucleic acid polymerase and primer extension with atemplate-dependent nucleic acid polymerase. The modification of theproducts may serve to add one or more labels or binding moieties, toalter mass, to add specific sequences, or to otherwise facilitatespecific analysis of the cleavage products.

Cleavage product may be used to complete a functional structure, such asa competent promoter for in vitro transcription or other protein bindingsite. The oligonucleotide product may also be used to complete acleavage structure to enable a subsequent invasive cleavage reaction,the product of which may be detected or used by any of the methodsdescribed above, including the participation in further invasivecleavage reactions.

It is envisioned that any or all of the oligonucleotide probes used inthe Invader™ assay may be made to contact non-contiguous sequences inthe target strand. In the Examples below, the upstream Invader™oligonucleotide is made to bridge a structure, thus directing thecleavage of a non-bridging probe.

Specific applications of the structure probing methods of the presentinvention are described below.

B. Detection and Identification of Pathogens Using the Structure ProbingMethod

1. Detection and Identification of Multi-Drug Resistant M. tuberculosis

In the past decade there has been a tremendous resurgence in theincidence of tuberculosis in this country and throughout the world. Inthe United States, the incidence of tuberculosis has risen steadilyduring past decade, accounting for 2000 deaths annually, with as many as10 million Americans infected with the disease. The situation iscritical in New York City, where the incidence has more than doubled inthe past decade, accounting for 14% of all new cases in the UnitedStates in 1990 (Frieden et al., New Engl. J. Med., 328:521 [1993]).

The crisis in New York City is particularly dire because a significantproportion (as many as one-third) of the recent cases are resistant toone or more anti-tuberculosis drugs (Frieden et al, supra and Hughes,Scrip Magazine May [1994]). Multi-drug resistant tuberculosis (MDR-TB)is an iatrogenic disease that arises from incomplete treatment of aprimary infection (Jacobs, Jr., Clin. Infect. Dis., 19:1 [1994]). MDR-TBappears to pose an especially serious risk to the immunocompromised, whoare more likely to be infected with MDR-TB strains than are otherwisehealthy individuals [Jacobs, Jr., supra]. The mortality rate of MDR-TBin immunocompromised individuals is alarmingly high, often exceeding90%, compared to a mortality rate of <50% in otherwise uncompromisedindividuals (Donnabella et al., Am. J. Respir. Dis., 11:639 [1994]).

From a clinical standpoint, tuberculosis has always been difficult todiagnose because of the extremely long generation time of Mycobacteriumtuberculosis as well as the environmental prevalence of other, fastergrowing mycobacterial species. The doubling time of M tuberculosis is20-24 hours, and growth by conventional methods typically requires 4 to6 weeks to positively identify M tuberculosis (Jacobs, Jr. et al.,Science 260:819 [1993] and Shinnick and Jones in Tuberculosis:Pathogenesis, Protection and Control, Bloom, ed., American Society ofMicrobiology, Washington, D.C. [1994], pp. 517-530). It can take anadditional 3 to 6 weeks to diagnose the drug susceptibility of a givenstrain (Shinnick and Jones, supra). Needless to say, the health risks tothe infected individual, as well as to the public, during a protractedperiod in which the patient may or may not be symptomatic, but is almostcertainly contagious, are considerable. Once a drug resistance profilehas been elucidated and a diagnosis made, treatment of a single patientcan cost up to $250,000 and require 24 months.

The recent explosion in the incidence of the disease, together with thedire risks posed by MDR strains, have combined to spur a burst ofresearch activity and commercial development of procedures and productsaimed at accelerating the detection of M tuberculosis as well theelucidation of drug resistance profiles of M tuberculosis clinicalisolates. A number of these methods are devoted primarily to the task ofdetermining whether a given strain is M tuberculosis or a mycobacterialspecies other than tuberculosis. Both culture based methods andnucleic-acid based methods have been developed that allow M tuberculosisto be positively identified more rapidly than by classical methods:detection times have been reduced from greater than 6 weeks to as littleas two weeks (culture-based methods) or two days (nucleic acid-basedmethods). While culture-based methods are currently in wide-spread usein clinical laboratories, a number of rapid nucleic acid-based methodsthat can be applied directly to clinical samples are under development.For all of the techniques described below, it is necessary to first“decontaminate” the clinical samples, such as sputum (usually done bypretreatment with N-acetyl L-cysteine and NaOH) to reduce contaminationby non-mycobacterial species (Shinnick and Jones, supra).

The polymerase chain reaction (PCR) has been applied to the detection ofM tuberculosis and can be used to detect its presence directly fromclinical specimens within one to two days. The more sensitive techniquesrely on a two-step procedure: the first step is the PCR amplificationitself, the second is an analytical step such as hybridization of theamplicon to a M tuberculosis-specific oligonucleotide probe, or analysisby RFLP or DNA sequencing (Shinnick and Jones, supra).

The Amplified M tuberculosis Direct Test (AMTDT; Gen-Probe) relies onTranscription Mediated Amplification (TMA; essentially a self-sustainedsequence reaction [3SR] amplification) to amplify target rRNA sequencesdirectly from clinical specimens. Once the rRNA has been amplified, itis then detected by a dye-labeled assay such as the PACE2. This assay ishighly subject to inhibition by substances present in clinical samples.

The Cycling Probe Reaction (CPR; ID Biomedical). This technique, whichis under development as a diagnostic tool for detecting the presence ofM tuberculosis, measures the accumulation of signal probe molecules. Thesignal amplification is accomplished by hybridizing tripartiteDNA-RNA-DNA probes to target nucleic acids, such as Mtuberculosis-specific sequences. Upon the addition of RNAse H, the RNAportion of the chimeric probe is degraded, releasing the DNA portions,which accumulate linearly over time to indicate that the target sequenceis present (Yule, Bio/Technol., 12:1335 [1994]). The need to use of RNAprobes is a drawback, particularly for use in crude clinical samples,where RNase contamination is often rampant.

The above nucleic acid-based detection and differentiation methods offera clear time savings over the more traditional, culture-based methods.While they are beginning to enter the clinical setting, their usefulnessin the routine diagnosis of M tuberculosis is still in question, inlarge part because of problems with associated with cross-contaminationand low-sensitivity relative to culture-based methods. In addition, manyof these procedures are limited to analysis of respiratory specimens(Yule, supra).

i) Determination of the Antibiotic Resistance Profile of M. tuberculosis

a) Culture-based methods: Once a positive identification of Mtuberculosis has been made, it is necessary to characterize the extentand nature of the strain's resistance to antibiotics. The traditionalmethod used to determine antibiotic resistance is the direct proportionagar dilution method, in which dilutions of culture are plated on mediacontaining antibiotics and on control media without antibiotics. Thismethod typically adds an additional 2-6 weeks to the time required fordiagnosis and characterization of an unknown clinical sample (Jacobs,Jr., supra).

The Luciferase Reporter Mycobacteriophage (LRM) assay was firstdescribed in 1993 (Jacobs, Jr. et al. [1993], supra). In this assay, amycobacteriophage containing a cloned copy of the luciferase gene isused to infect mycobacterial cultures. In the presence of luciferin andATP, the expressed luciferase produces photons, easily distinguishableby eye or by a luminometer, allowing a precise determination of theextent of mycobacterial growth in the presence of antibiotics. Oncesufficient culture has been obtained (usually 10-14 dayspost-inoculation), the assay can be completed in 2 days. This methodsuffers from the fact that the LRM are not specific for M tuberculosis:they also infect M. smegmatis and M bovis (e.g., BCG), therebycomplicating the interpretation of positive results. Discriminationbetween the two species must be accomplished by growth on specializedmedia which does not support the growth of M. tuberculosis (e.g., NAPmedia). This confirmation requires another 2 to 4 days.

The above culture-based methods for determining antibiotic resistancewill continue to play a role in assessing the effectiveness of putativenew anti-mycobacterial agents and those drugs for which a genetic targethas not yet been identified. However, recent success in elucidating themolecular basis for resistance to a number of anti-mycobacterial agents,including many of the front-line drugs, has made possible the use ofmuch faster, more accurate and more informative DNA polymorphism-basedassays.

b) DNA-based methods: Genetic loci involved in resistance to isoniazid,rifampin, streptomycin, fluoroquinolones, and ethionamide have beenidentified (Jacobs, Jr., supra; Heym et al., Lancet 344:293 [1994]; andMorris et al., J. Infect. Dis., 171:954 [1995]. A combination ofisoniazid (inh) and rifampin (rif) along with pyrazinamide andethambutol or streptomycin, is routinely used as the first line ofattack against confirmed cases of M tuberculosis (Banerjee et al.,Science 263:227 [1994]). Consequently, resistance to one or more ofthese drugs can have disastrous implications for short coursechemotherapy treatment. The increasing incidence of such resistantstrains necessitates the development of rapid assays to detect them andthereby reduce the expense and community health hazards of pursuingineffective, and possibly detrimental, treatments. The identification ofsome of the genetic loci involved in drug resistance has facilitated theadoption of mutation detection technologies for rapid screening ofnucleotide changes that result in drug resistance. The availability ofamplification procedures such as PCR and SDA, which have been successfulin replicating large amounts of target DNA directly from clinicalspecimens, makes DNA-based approaches to antibiotic profiling far morerapid than conventional, culture-based methods.

The most widely employed techniques in the genetic identification ofmutations leading to drug resistance are DNA sequencing, RestrictionFragment Length Polymorphism (RFLP), PCR-Single Stranded ConformationalPolymorphism (PCR-SSCP), and PCR-dideoxyfingerprinting (PCR-ddF). All ofthese techniques have drawbacks as discussed above. None of them offersa rapid, reproducible means of precisely and uniquely identifyingindividual alleles.

In contrast, the structure probing methods of the present inventionprovide an approach that relies on interactions of oligonucleotideprobes with the target nucleic acid on the primary, secondary andtertiary structure level. This method requires a fraction of the time,skill and expense of the techniques described above, and can beperformed using instrumentation commonly found in the clinical lab(e.g., a microtiter plate reader).

The application of this method to the detection of MDR-TB is illustratedherein using segments of DNA amplified from katG gene. Other genesassociated with MDR-TB, including but not limited to those involved inconferring resistance to isoniazid (inhA), streptomycin (rpsL and rrs),and fluoroquinoline (gyrA), are equally well suited to the structureprobing assay of the present invention.

2. Detection and Identification of Hepatitis C Virus

Hepatitis C virus (HCV) infection is the predominant cause ofpost-transfusion non-A, non-B (NANB) hepatitis around the world. Inaddition, HCV is the major etiologic agent of hepatocellular carcinoma(HCC) and chronic liver disease world wide. HCV infection is transmittedprimarily to blood transfusion recipients and intravenous drug usersalthough maternal transmission to offspring and transmission torecipients of organ transplants have been reported.

The genome of the positive-stranded RNA hepatitis C virus comprisesseveral regions including 5′ and 3′ noncoding regions (i.e., 5′ and 3′untranslated regions) and a polyprotein coding region which encodes thecore protein (C), two envelope glycoproteins (E1 and E2/NS1) and sixnonstructural glycoproteins (NS2-NS5b). Molecular biological analysis ofthe small (9.4 kb) RNA genome has showed that some regions of the genomeare very highly conserved between isolates, while other regions arefairly rapidly changeable. The 5′ noncoding region (NCR) is the mosthighly conserved region in the HCV. These analyses have allowed theseviruses to be divided into six basic genotype groups, and then furtherclassified into over a dozen sub-types (the nomenclature and division ofHCV genotypes is evolving; See Altamirano et al., J. Infect. Dis.,171:1034 [1995] for a recent classification scheme). These viral groupsare associated with different geographical areas, and accurateidentification of the agent in outbreaks is important in monitoring thedisease. While only Group 1 HCV has been observed in the United States,multiple HCV genotypes have been observed in both Europe and Japan.

The ability to determine the genotype of viral isolates also allowscomparisons of the clinical outcomes from infection by the differenttypes of HCV, and from infection by multiple types in a singleindividual. HCV type has also been associated with differential efficacyof treatment with interferon, with Group 1 infected individuals showinglittle response (Kanai et al., Lancet 339:1543 [1992] and Yoshioka etal., Hepatol., 16:293 [1992]). Pre-screening of infected individuals forthe viral type will allow the clinician to make a more accuratediagnosis, and to avoid costly but fruitless drug treatment.

Existing methods for determining the genotype of HCV isolates includetraditional serotyping, PCR amplification of segments of the HCV genomecoupled with either DNA sequencing or hybridization to HCV-specificprobes and RFLP analysis of PCR amplified HCV DNA. All of these methodssuffer from the limitations discussed above (i.e., DNA sequencing is toolabor-intensive and expensive to be practical in clinical laboratorysettings; RFLP analysis suffers from low sensitivity).

Universal and genotype specific primers have been designed for theamplification of HCV sequences from RNA extracted from plasma or serum(Okamoto et al., J. Gen. Virol., 73:673 [1992]; Yoshioka et al.,Hepatol., 16:293 [1992] and Altamirano et al., supra). These primers canbe used to generate PCR products which serve as substrates in thestructure probing assay of the present invention. As shown herein, thestructure probing assay provides a rapid and accurate method of typingHCV isolates. The structure probing analysis of HCV substrates allows adistinction to be made between the major genotypes and subtypes of HCVthus providing improved methods for the genotyping of HCV isolates.

3. Detection and Identification of Bacterial Pathogens

Identification and typing of bacterial pathogens is critical in theclinical management of infectious diseases. Precise identity of amicrobe is used not only to differentiate a disease state from a healthystate, but is also fundamental to determining whether and whichantibiotics or other antimicrobial therapies are most suitable fortreatment. Traditional methods of pathogen typing have used a variety ofphenotypic features, including growth characteristics, color, cell orcolony morphology, antibiotic susceptibility, staining, smell andreactivity with specific antibodies to identify bacteria. All of thesemethods require culture of the suspected pathogen, which suffers from anumber of serious shortcomings, including high material and labor costs,danger of worker exposure, false positives due to mishandling and falsenegatives due to low numbers of viable cells or due to the fastidiousculture requirements of many pathogens. In addition, culture methodsrequire a relatively long time to achieve diagnosis, and because of thepotentially life-threatening nature of such infections, antimicrobialtherapy is often started before the results can be obtained. In manycases the pathogens are very similar to the organisms that make up thenormal flora, and may be indistinguishable from the innocuous strains bythe methods cited above. In these cases, determination of the presenceof the pathogenic strain may require the higher resolution afforded bymore recently developed molecular typing methods.

A number of methods of examining the genetic material from organisms ofinterest have been developed. One way of performing this type ofanalysis is by hybridization of species-specific nucleic acid probes tothe DNA or RNA from the organism to be tested. This is done byimmobilizing the denatured nucleic acid to be tested on a membranesupport, and probing with labeled nucleic acids that will bind only inthe presence of the DNA or RNA from the pathogen. In this way, pathogenscan be identified. Organisms can be further differentiated by using theRFLP method described above, in which the genomic DNA is digested withone or more restriction enzymes before electrophoretic separation andtransfer to a nitrocellulose or nylon membrane support. Probing with thespecies-specific nucleic acid probes will reveal a banding pattern that,if it shows variation between isolates, can be used as a reproducibleway of discriminating between strains. However, these methods aresusceptible to the drawbacks outlined above: assays based onsequence-specific hybridization to complex (i.e., whole genome) targetsare time-consuming and may give false or misleading results if thestringency of the hybridization is not well controlled, and RFLPidentification is dependent on the presence of suitable restrictionsites in the DNA to be analyzed.

To address these concerns about hybridization and RFLP as diagnostictools, several methods of molecular analysis based on polymerase chainreaction (PCR) amplification have gained popularity. In onewell-accepted method, called PCR fingerprinting, the size of a fragmentgenerated by PCR is used as an identifier. In this type of assay, theprimers are targeted to regions containing variable numbers of tandemrepeated sequences (referred to as VNTRs an eukaryotes). The number ofrepeats, and thus the length of the PCR amplicon, can be characteristicof a given pathogen, and co-amplification of several of these loci in asingle reaction can create specific and reproducible fingerprints,allowing discrimination between closely related species.

In some cases where organisms are very closely related, however, thetarget of the amplification does not display a size difference, and theamplified segment must be further probed to achieve more preciseidentification. This may be done on a solid support, in a fashionanalogous to the whole-genome hybridization described above, but thishas the same problem with variable stringency as that assay.Alternatively, the interior of the PCR fragment may be used as atemplate for a sequence-specific ligation event. As outlined above forthe LCR, in this method, single stranded probes to be ligated arepositioned along the sequence of interest on either side of anidentifying polymorphism, so that the success or failure of the ligationwill indicate the presence or absence of a specific nucleotide sequenceat that site. With either hybridization or ligation methods of PCRproduct analysis, knowledge of the precise sequence in the area of probebinding must be obtained in advance, and differences outside the probebinding area are not detected. These methods are poorly suited to theexamination and typing of new isolates that have not been fullycharacterized.

In the methods of the present invention, primers that recognizeconserved regions of bacterial ribosomal RNA genes allow amplificationof segments of these genes that include sites of variation. Thevariations in ribosomal gene sequences have become an accepted methodnot only of differentiating between similar organisms on a DNA sequencelevel, but their consistent rate of change allows these sequences to beused to evaluate the evolutionary relatedness of organisms. That is tosay, the more similar the nucleic acid is at the sequence level, themore closely related the organisms in discussion are considered to be(Woese, Microbiol. Rev., 51:221-271 [1987]). The present inventionallows the amplification products derived from these sequences to beused to create highly individual structural fingerprints (e.g., profilesof the complex formation with an array of probes), allowing thedetection of sequence polymorphisms without prior knowledge of the site,character or even the presence of the polymorphisms. With appropriateselection of primers, the PCR amplification can be made to be eitherall-inclusive (e.g., using the most highly conserved ribosomalsequences) to generate PCR products that, when analyzed using themethods of the present invention, allow comparison of distantly relatedorganisms, or the primers can be chosen to be very specific for a givengenus, to allow examination at the species and subspecies level. Whilethe examination of ribosomal genes is extremely useful in thesecharacterizations, the use of the structure probing method in bacterialtyping is not limited to these genes. Other genes, including but notlimited to those associated with specific growth characteristics, (e.g.,carbon source preference, antibiotic resistance, resistance tomethicillin or antigen production), or with particular cell morphologies(such as pilus formation) are equally well suited to the structureprobing assay of the present invention.

C. Extraction of Nucleic Acids From Clinical Samples

To provide nucleic acid substrates for use in the detection andidentification of microorganisms in clinical samples using the structureprobing assay, nucleic acid is extracted from the sample. The nucleicacid may be extracted from a variety of clinical samples (fresh orfrozen tissue, suspensions of cells [e.g., blood], cerebral spinalfluid, sputum, urine, etc.) using a variety of standard techniques orcommercially available kits. For example, kits which allow the isolationof RNA or DNA from tissue samples are available from Qiagen, Inc.(Chatsworth, Calif.) and Stratagene (La Jolla, Calif.). For example, theQIAamp Blood kits permit the isolation of DNA from blood (fresh, frozenor dried) as well as bone marrow, body fluids or cell suspensions.QIAamp tissue kits permit the isolation of DNA from tissues such asmuscles, organs and tumors.

It has been found that crude extracts from relatively homogenousspecimens (such as blood, bacterial colonies, viral plaques, or cerebralspinal fluid) are better suited to severing as templates for theamplification of unique PCR products than are more composite specimens(such as urine, sputum or feces) (Shibata in PCR. The Polymerase ChainReaction, Mullis et al., eds., Birkhauser, Boston [1994], pp. 47-54).Samples which contain relatively few copies of the material to beamplified (i.e., the target nucleic acid), such as cerebral spinalfluid, can be added directly to a PCR. Blood samples have posed aspecial problem in PCRs due to the inhibitory properties of red bloodcells. The red blood cells must be removed prior to the use of blood ina PCR; there are both classical and commercially available methods forthis purpose (e.g., QIAamp Blood kits, passage through a Chelex 100column [BioRad], etc.). Extraction of nucleic acid from sputum, thespecimen of choice for the direct detection of M. tuberculosis, requiresprior decontamination to kill or inhibit the growth of other bacterialspecies. This decontamination is typically accomplished by treatment ofthe sample with N-acetyl L-cysteine and NaOH (Shinnick and Jones,supra). This decontamination process is necessary only when the sputumspecimen is to be cultured prior to analysis.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the disclosure which follows, the following abbreviations apply: ° C.(degrees Centigrade); g (gravitational field); vol (volume); w/v (weightto volume); v/v (volume to volume); BSA (bovine serum albumin); CTAB(cetyltrimethylammuonium bromide); HPLC (high pressure liquidchromatography); DNA (deoxyribonucleic acid); IVS (interveningsequence); p (plasmid); ml (microliters); ml (milliliters); mg(micrograms); pmoles (picomoles); mg (milligrams); MOPS(3-[N-Morpholino]propanesulfonic acid); M (molar); mM (milliMolar); mM(microMolar); nm (nanometers); nt (nucleotide); bp (base pair); kb(kilobase pair); kdal (kilodaltons); OD (optical density); EDTA(ethylene diamine tetra-acetic acid); FITC (fluorescein isothiocyanate);IPTG (isopropylthiogalactoside); X-Gal(5-bromo-4-chloro-3-indolyl-b-D-galactosidase); SDS (sodium dodecylsulfate); NaPO₄ (sodium phosphate); Tris(tris(hydroxymethyl)-aminomethane); PMSF(phenylmethyl-sulfonylfluoride); TBE (Tris-Borate-EDTA, i.e., Trisbuffer titrated with boric acid rather than HCl and containing EDTA);PBS (phosphate buffered saline); Ab Peptides (Ab Peptides, St. Louis,Mo.); PPBS (phosphate buffered saline containing 1 mM PMSF); PAGE(polyacrylamide gel electrophoresis); Tween (polyoxyethylene-sorbitan);JBL (JBL, San Louis Obispo, Calif.); Boehringer Mannheim (BoehringerMannheim, Indianapolis, Ind.); Dynal (Dynal A.S., Oslo, Norway);Epicentre (Epicentre Technologies, Madison, Wis.); MJ Research (MJResearch, Inc., Watertown, Mass.); National Biosciences (NationalBiosciences, Plymouth, MN); New England Biolabs (New England Biolabs,Beverly, Mass.); Novagen (Novagen, Inc., Madison, Wis.); Perkin Elmer(Perkin Elmer, Norwalk, Conn.); Promega Corp. (Promega Corp., Madison,Wis.); Stratagene (Stratagene Cloning Systems, La Jolla, Calif.); Third-Wave (Third Wave Technologies, Inc., Madison, Wis.); and USB (U.S.Biochemical, Cleveland, Ohio).

20× SSPE (sodium chloride, sodium phosphate, EDTA) contains per liter:174 grams NaCl, 27.6 grams NaH₂PO₄.H₂O and 7.4 grams EDTA; the pH isadjusted to 7.4 with NaOH. PBS (phosphate-buffered saline) contains perliter: 8 grams NaCl, 0.2 grams KCl, 1.44 grams Na₂PO₄ and 0.24 gramsKH₂PO₄; the pH is adjusted to 7.4 with HCl.

EXAMPLE 1

The Presence of a Structure and a Probe Mismatch in Combination ProvideMore Sensitive Discrimination Than Does Either Effect Alone

In this Example, the effects on oligonucleotide binding of either theformation of an occlusive structure, the presence of a single-basemismatch, or the presence of both at once were examined. To separate theeffects on the efficiency of binding of structure from the effects ofmismatches, four katG DNA target variants were chosen (SEQ ID NOS:1, 2,3 and 4). The structures of these four targets in the region of theprobe hybridization sites are shown in FIGS. 2A-2D and the existence ofthe large stem-loop in structures 2C. and 2D (SEQ ID NOS:3 and 4,respectively) was confirmed by digestion with the structure-specificCleavase®I nuclease (Third Wave) and the cleavage sites are indicated bythe arrows on structures 2C and 2D. The dark bar on the left of eachstructure in FIGS. 2A-2D indicates the region to which the capture probeis expected to bind. The pointed kink in the black bar in structures 2Band 2D indicates a site of mismatch between the capture probe and thekatG target.

a) CFLP® Analysis of Mutations in the katG Gene of M. tuberculosis

i) Generation of Plasmids Containing katG Gene Sequences

Genomic DNA isolated from wild-type M tuberculosis or M tuberculosisstrains containing mutations in the katG gene associated with isoniazidresistance were obtained from Dr. J. Uhl (Mayo Clinic, Rochester,Minn.). These strains are termed wild-type and S315T (Cockerill, III etal., J. Infect. Dis., 171:240 [1995]). Strain S315T contains a G to Cmutation in codon 315 of the wild-type katG gene.

A 620 bp region of the M tuberculosis katG gene was amplified using thePCR from DNA derived from the above strains. The primers used to amplifythe katG gene sequences were KatG904 (5′-AGCTCGTATGGCACCGGAAC-3′)(SEQ IDNO:5) and KatG1523 (5′-TTGACCTCCCACCCGACTTG-3′) (SEQ ID NO:6); theseprimers amplify a 620 bp region of katG gene. The PCRs were conducted ina final reaction volume of 100 μl and contained the KatG904 and KatG1523primers at 0.5 mM, 1.5 mM MgCl₂, 20 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.05% each Tween®-20 and Nonidet® P-40 non-ionic detergents, 60 mM of all 4dNTPs.

The reaction mixtures were heated at 95° C. for 3 min, thenamplification was started with addition of 5 units of Taq DNA polymeraseand continued for 35 cycles at 95° C. for 1 min, 60° C. for 1 min and72° C. for 2 min.

To clone the PCR-amplified katG fragments, 1 μl of each PCR product wasused for ligation into the linear pT7Blue T-vector (Novagen, Madison,Wis.). The ligation products were used to transform competent JM 109cells and clones containing pT7Blue T-vector with an insert wereselected by white color on LB plates containing 40 mg/ml X-Gal, 40 mg/mlIPTG and 50 mg/ml ampicillin. For each of the PCR samples, four colonieswere picked and grown overnight in 2 ml LB media containing 50 mg/mlcarbenicillin. Plasmid DNA was isolated using an alkaline miniprepprotocol (Sambrook et al., supra).

To analyze the cloned katG fragments, 1 μl of plasmid DNA from eachclone was amplified by PCR using 100 μl reactions containing the KatG904and KatG1523 primers at 0.5 mM, 1.5 mM MgCl₂, 20 mM Tris-HCl, pH 8.3, 50mM KCl, 0.05 % each Tween®-20 and Nonidet® P-40 non-ionic detergents, 60mM of all 4 dNTPs and 5 units of Taq DNA polymerase. The PCRs werecycled 35 times at 95° C. for 1 min, 60° C. for 1 min and 72° C. for 2min. PCR products were separated by electrophoresis on a 6% nativepolyacrylamide gel in 0.5× TBE buffer and clones that gave rise to a 620bp fragment were selected for further analysis.

Fragments of DNA (391 bp), labeled on the 5′ end of the sense strandwith tetrachlorofluorescein (TET), were created from the cloned katGgenes using primers 5′-TET-AGCTCGTATGGCACCGGAACC-3′ (SEQ ID NO:7) and5′-GGACCAGC GGCCCAAGGTAT-3′ (SEQ ID NO:8). When the wild type katG DNAfragment of this size is denatured by heating and allowed to fold,nucleotides A37-C45 base pair with nucleotides G381-T389 (measured fromthe 5′ end of the sense strand). The wild type sequence has a G at bp 41(G41) which is complimentary to the C at bp 385 (C385) as shown in FIG.2C; the S315T mutant sequence contains a C at bp 41 (C41) which isnon-complimentary to C385 and disrupts the formation of the hairpin, asshown in FIG. 2B. Two additional non-wild type sequences were created byusing an alternative primer at the 3′ end (5′-GGACCACCGGCCCAAGGTATCT-3′;SEQ ID NO:9) which changed C385 to G385. This allowed creation offragments with a G41 to G385 mismatch (FIG. 2A) and a C41 to G385 basepair (FIG. 2D).

The PCR reactions were performed as follows: PCR mixtures contained 5 ngof plasmid DNA template, 1× PCR buffer, 200 mM of each dNTP, 0.5 mM ofeach primer, 5 units of Taq Polymerase and water to final volume of 100ml. The PCR cycling conditions were: 95° C. for 45 ″, 65° C. for 1′30″and 72° C. for 2′ for a total of 30 cycles, followed by a 4° C. soak.The 391 bp PCR products were purified using “High Pure PCR ProductPurification Kit” (Boehringer Mannheim). This set of fragments (SEQ IDNOS:1-4) allowed a single probe to be used to assess the effects ofmismatch, secondary structure or a combination of both on the formationof the complex between the probe and target.

ii) CFLP® Reactions

CFLP® reactions were performed on each 5′-TET labeled amplificationproduct from the four KatG variants (2A-2D). Each CFLP® reactioncontained approximately 20 fmole of the amplified product, 50 units ofCleavase® I nuclease in 10 μl of 1× CFLP® buffer (10 mM MOPS pH 7.5,0.05% Tween® 20 and 0.05% Nonidet® P40 non-ionic detergents) with 0.2 mMMnCl₂. Reactions were assembled with all components except the enzymeand the MnCl₂, heated to 95° C. for 15 seconds, then cooled to thereaction temperature of 50° C. The cleavage reactions were started withthe addition of the enzyme and the MnCl₂, and incubated for 5 minutes.The reactions were terminated by the addition of 4 ml of 95% formamidewith 10 mM EDTA and 0.02% Methyl Violet. The products were heated at 95°C. for 30 sec, and aliquots were resolved by electrophoresis through 10%denaturing polyacrylamide gel (19:1 cross link) with 7 M urea in abuffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. The gel was visualizedusing the FMBIO-100 Image Analyzer (Hitachi). The resulting image isshown in the left panel of FIG. 3. Lanes A-D contain CFLP reactionproducts from reactions containing structures 2A-2D, respectively. LanesC and D contain a product (37 nt; indicated by the arrowhead) notpresent in lanes A and B which indicates the presence of the largestem-loop in structures 2C and 2D shown, respectively in FIGS. 2C and2D.

b) Structure Probing Analysis of M. tuberculosis katG Gene Targets

In these experiments, the capture probes are bound to the target DNAs insolution and then immobilized on a solid support. The 391 bp fragment ofkatG described above was created by PCR using a 5′-fluorescein labelledprimer (SEQ ID NO:7). A hybridization mixture was assembled, containing40 fmoles of heat-denatured, 391 bp katG PCR product having one of thefour sequences depicted in FIGS. 2A-2D (SEQ ID NOS:1-4), labelled on the5′ end of the sense strand, 1.5 pmole of the biotinylated capture probe(SEQ ID NO:10), 0.01 mg/ml tRNA, 0.2% acetylated BSA, 4.5× SSPE and H2to 150 μl.

Aliquots (100 μl) of the mixture were then transferred to wells in astreptavidin-coated 96-well plate (Boehringer Mannheim) and incubated atroom temperature for 30 min. The plate was then washed three times with1× PBS, with 0.01% Tween®-20 non-ionic detergent, then treated with asolution containing 0.2% 1-Block (Tropix, Bedford, Mass.) and 0.05%Tween®-20 non-ionic detergent in PBS for 30 minutes to block. Afterblocking, the plate was washed three times with PBS with 0.1% Tween®-20non-ionic detergent. A 1:5000 dilution of 0.75 u/ml anti-fluoresceinantibody conjugated with alkaline-phosphatase in 0.2% I-block buffer wasadded to the plate in 100 μl/well volumes. After ½ hour, the plate waswashed three times with TBS (25 mM Tris-Cl, 0.15 M NaCl, pH 7.2). Onehundred microliters of Attophos® fluorescent substrate (JBL) was addedto each well and the plate was incubated at room temperature for 1 hourbefore fluorescence readings were taken using a Perkin-ElmerCytofluor-4000 set to excite at 450/50 nm and to and detect emission at580/50 nm. Each assay was performed in triplicate and the standarddeviation is represented by the black bar at the top of each column inthe right panel of FIG. 3. The fluorescence intensity is indicated inarbitrary fluorescence units. In FIG. 3, “A-D” indicates the use ofstructures 2A-2D, respectively in the structure probing assay.

The results, shown in FIG. 3, indicate that not only the mismatchbetween target DNA and probe, but also differences in secondarystructure, leads to a better discrimination between wild type and mutantDNA.

EXAMPLE 2 Changes in DNA Secondary Structure Leads to Different BindingAbilities Between the Target DNA and the Capture Probe

The context of a target sequence (i.e., the length and identity of theflanking nucleic acid), can influence the secondary structure, andtherefore the hybridization accessibility of the target segment. Toillustrate this effect, a target segment of DNA was exposed, either withor without pretreatment with a restriction enzyme, to a capture probethat is complementary to a site that is unaffected by the restrictioncleavage. The restriction enzyme BamHI was used to digest the 391 bp5′-fluorescein labeled fragments of katG DNA, either wild-type (FIG. 2C)or the S315T mutant (FIG. 2B), prepared as described in Example 1. Therestriction enzyme shortens the 5′ labelled fragment from 391 nt to 256nt. The capture probe is complementary to sequence located within thefirst 50 nt of these katG DNA targets. Equal amounts of the DNA targetswere used in all the reactions. The restriction digests included 2pmoles of 5′-Fluorescein labeled DNA, 10 μl of 10× BamHI buffer, 160units of BamHI enzyme and H₂O to a final volume of 100 μl. The reactionswere incubated at 37° C. for 2 hours. After digestion, the hybridizationassay was performed as described above, using the capture probe (SEQ IDNO:10). The results are shown in FIG. 4. In FIG. 4, the amount oflabeled target captured (as a target/probe complex) is shown for eachtarget/probe complex examined (shown using arbitrary fluorescenceunits). In FIG. 4, the following abbreviations are used: C (structure2C); B (structure 2B); C/BamHI (BamHI-digested structure 2C); B/BamHI(BamHI-digested structure 2B).

The 2C DNA target (SEQ ID NO:3) has a site perfectly complementary tothe capture probe, while the 2B DNA target (SEQ ID NO:2) has a singlebase mismatch near the middle of the region of complementarity with thecapture probe. Despite this mismatch, discrimination between these two391 nt DNAs (i.e., not digested with BamHI) by hybridization to thisprobe is very weak. As shown in FIG. 4, the difference in the bindingefficiency between wild type and mutant DNA after enzyme digestion isincreased. Because the segment of the katG DNA to which the probehybridizes is not cleaved by the enzyme, it can be concluded that it isthe change in the folded structure of the target DNA that accounts forthe change in the hybridization pattern. This shows that, whilemismatches may enhance discrimination between nucleic acid variants,they are not necessary for discrimination between DNAs by hybridization.These results also demonstrate that variables other than the degree ofcomplementarity (e.g., complete or partial) between the probe and target(e.g., the secondary and tertiary structure of the target) may provide abetter means of discriminating between related sequences.

EXAMPLE 3 Hybridization Analysis Using Multiple Capture Probes for HCVGenotyping

Because both mismatches and structures are used in the method of thepresent invention for discrimination between similar nucleic acids byhybridization, the patterns created by the use of a structure specificnuclease, e.g., Cleavase® I nuclease can be used as a way of selectingregions likely to demonstrate different binding behaviors with differentvariants. Because the CFLP® method indicates the presence of structurein a DNA fragment of interest, and because the variations in thestructures tend to be proximal to the actual sequence changes, choosingcapture probes at or near the CFLP® cleavage sites increases theprobability of choosing a sequence that changes in accessibility in thedifferent variants. FIG. 5 shows a diagram depicting this means of probeselection as applied to the comparison of fragments from the Hepatitis Cvirus. In FIG. 5, the left panel shows an fluoroimager scan ofsequencing gel in which products of CFLP® cleavage reactions areresolved next to a sequencing ladder generated using the same target DNAemployed in the CFLP® cleavage reactions. The middle panel provides anenlargement of sections of the gel shown in the left panel. The rightpanel provides the sequence of nine HCV probes (SEQ ID NOS:11-19); theseprobe were synthesized such that they contained a 5′-biotin moiety.

Five subtypes of HCV; 1a, 1b, 2b, 2c, and 3a were analyzed using boththe CFLP® cleavage method, and cycle sequencing. The CFLP® reactionswere performed on each 5′-fluorescein labeled amplification product fromeach HCV isolate as follows. Each CFLP® reaction contained approximately20 fmole of the amplified product, 25 units of Cleavase® I nuclease in10 μl of 1× CFLP® buffer (10 mM MOPS pH 7.5, 0.05% Tween® 20 and 0.05%Nonidet® P40 non-ionic detergents) with 0.2 mM MnCl₂. Reactions wereassembled with all components except the enzyme and the MnCl₂, heated to95° C. for 15 seconds, then cooled to the reaction temperature of 55° C.The cleavage reactions were started with the addition of the enzyme andthe MnCl₂, and incubated for 2 minutes. The reactions were terminated bythe addition of 4 μl of 95% formamide with 10 mM EDTA and 0.02% MethylViolet. The products were heated at 85° C. for 2 min, and aliquots wereresolved by electrophoresis through 10% denaturing polyacrylamide gel(19:1 cross link) with 7 M urea in a buffer of 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA. The gel was visualized using the FMBIO-100 ImageAnalyzer (Hitachi).

The CFLP® patterns for these HCV subtypes are shown in FIG. 5. Differentsubtypes of HCV give different CFLP® patterns, which means that theyalso have different internal secondary structure. Probes were designedto detect structure differences between the 1a, 1b, 2c and 3a HCVsubtypes. The capture probes are shown in the right panel of FIG. 5. Theregion to which each of these HCV capture probes can bind along thesequence of the HCV targets is shown in FIG. 6. In FIG. 6, the locationof the probe binding regions are indicated using bold type, underliningand by placing the probe designation above the sequence. The consensusHCV sequence (SEQ ID NO:20), and the sequence of HCV subtypes 1a, 1b, 2cand 3a (SEQ ID NOS:20-23, respectively) are provided.

The capture probes (SEQ ID NOS: 11-19) were synthetically labeled withbiotin at their 5′ end and purified by gel-electrophoresis. The HCVtarget DNA was labeled with fluorescein at the 5′ end of the antisensestrand by PCR using a 5′- fluorescein labeled primer. The primersemployed for the amplification of HCV target DNAs were: 5′ primer:5′-Fl-CTCGCAAGCACCCTATCA (SEQ ID NO:24) and 3′ primer:5′-GCAGAAAGCGTCTAGCCATGG (SEQ ID NO:25). The PCR reactions included 5 ngof plasmid DNA template, 1× PCR buffer (Boehringer Mannheim), 200 mM ofeach dNTP, 0.5 mM of each primer (SEQ ID NOS:24 and 25), 5 units Taq DNApolymerase (Boehringer Mannheim) and water to a final volume of 100 μl.The PCR cycling conditions were: 95° C. for 45″, 55° C. for 45″, and 72°C. for 1′, for 30 cycles followed by a 72° C. for 5′ extension and a 4°C. soak. The resulting 244 bp PCR products (SEQ ID NOS:26-29 for types1a, 1b, 2c and 3a, respectively) were purified using “High Pure PCRProduct Purification Kit” (Boehringer Mannheim) and eluted in dH₂Oaccording to the manufacturer's instructions. The same amount of DNA,based on optical absorbance, was used for each sample in the captureassay. Structure probing analysis on streptavidin-coated 96-wellmicro-titer plates was performed as described above. Each assay wasperformed in triplicate and the standard deviation is shown as a blackbar at the top of each column in FIG. 7. The results are shown in FIG.7.

The column graphs of the measured fluorescence intensity for thecomplexes between each probe and a given target constitute acharacteristic “signature” that is distinctive for each HCV subtype. Theeffects of structure can be illustrated by examining the signalstrengths from targets binding to probe #40 (SEQ ID NO:16). While boththe 1b and 3a targets are completely complementary to probe #40, the 3atarget shows nearly undetectable signal, while the type 1b target signalis very strong. The binding of probe #251 (SEQ ID NO:12) to the HCVtargets shows similar signal variation even though this probe iscompletely complementary to all four of the HCV subtype targets.

EXAMPLE 4 Effect of Temperature on Structure Probing WithOligonucleotides

Most traditional hybridization methods have a small window oftemperature (i.e., about less than 10° C.) in which to produce theexpected discrimination between targets. The structure probing analysisof the four HCV subtypes (describe above) under different hybridizationtemperatures was performed to examine the effect of temperature on boththe secondary structure of DNA and the stability of the probe/targetcomplex. Three different temperatures were used; room temperature(approx. 20 to 25° C.), 37° C. and 50° C.

The profile of the HCV subtypes 1a, 1b and 3a are shown in FIG. 7. Theprofiles of the HCV subtype 1b are shown in FIG. 8B. The profiles of theHCV subtype 3a are shown in FIG. 8C. The hybridization profiles of thesethree HCV subtypes over a 25° C. range of temperature (˜25-50° C.) areshown in FIGS. 8A-8C (the numbers below each column indicates thecapture probe employed; note the change in scale for each temperaturetested). The profiles for these three HCV subtypes are essentially thesame over the 25° C. range of temperature tested. However, the higherthe temperature employed, the less stable the probe-DNA target bindingbecomes, so the overall fluorescence intensity was reduced. Theseresults show that the discrimination capability of the structure probingmethod is very robust, maintaining consistency over a broad range oftemperature.

EXAMPLE 5 Structure Probing Analysis of HCV Clinical Isolates

Structure probing analysis of HCV clinical isolates at a roomtemperature hybridization temperature was performed to examine thefeasibility of developing a diagnostic test for HCV genotyping. TwelveHCV amplification products generated from clinical samples were obtained(Molecular Pathology Dept, Univ. of Wisconsin Clinics, Madison, Wis.)and employed in the structure probe assay. These targets were RT-PCRproducts of viral RNA from different patient samples amplified using theAmplicor HCV detection kit (Roche Molecular Systems, Alameda, Calif.).Further PCR reactions were performed on these clinical amplificationproducts using the primer pair described in Example 4 (SEQ ID NOS:24 and25) to create ds PCR products comprising 5′ fluorescein labels on theanti-sense strands. The PCR conditions were as described in Example 4.The resulting HCV targets were employed in the structure probing assaywhich was carried out as described in Example 1.

The resulting profiles were sorted by type (based on the profilesdetermined for the HCV subtypes as described in Examples 3 and 4 andFIG. 7) and are shown in FIGS. 9A-9D (the types were independentlydetermined by single pass DNA sequencing. The resulting partialsequences, sufficient to identify types are as follows: #67 (SEQ IDNO:30), #69 (SEQ ID NO:31), #72 (SEQ ID NO:32), #73 (SEQ ID NO:33), #74(SEQ ID NO:34), #81 (SEQ ID NO:35), #85 (SEQ ID NO:36), #86 (SEQ IDNO:37) and #91 (SEQ ID NO:38).

The profiles for four different amplicons of HCV type 1a are shown inFIG. 9A (#69, #72, #73 and #85) and all have a profile similar to thetype 1a profile shown in FIG. 7. The profiles of three differentamplicons of HCV type 3a are shown in FIG. 9B (#81, #91 and #95) andtheir profiles are all similar to each other and to the type 3a profileshown in FIG. 7. The profile of an amplicon of HCV type 2c (#67) and anamplicon of HCV type 2b (#74) are shown in FIG. 9D. The profiles for twoamplicons of HCV 1b are shown in FIG. 9C (#66 and #86).

The profile for amplicon #86 was more similar to that of type 1a ratherthan type lb. Based on CFLP® analysis, amplicon #86 was classified astype 1b. However, using the probe set shown in FIG. 9C, thehybridization profile obtained in the structure probing assay appearedmore similar to that of type 1a. Sequence analysis showed that there isan extra mutation in this sample, which changed its hybridizationresponse to probe #40, creating a profile more like that of type 1a.Based on this T to C mutation in amplicon #86, an additional captureprobe having a sequence completely complimentary to amplicon #86 wastested (probe #53; SEQ ID NO:19). A structure probing assay using theamplicon #86 target and capture probe #53 generated a profile similar toa more typical type 1b profile. These results demonstrate thatadditional information concerning the structure of the amplicon #86target was obtained using the structure probing assay.

These data demonstrate that an unknown (i.e., uncharacterized) set ofHCV isolates can be identified by HCV type through the use of thestructure probing assay, with comparison of the resulting profiles tothose of previously characterized isolates (i.e., reference profiles).

It is clear from the above that the present invention provides methodsfor the analysis of the characteristic conformations of nucleic acidswithout the need for either electrophoretic separation of conformationsor fragments or for elaborate and expensive methods of visualizing gels(e.g., darkroom supplies, blotting equipment or fluorescence imagers).The novel methods of the present invention allow the rapididentification of variants (e.g., mutations) within human genes as wellas the detection and identification of pathogens in clinical samples.

Thus, the previous Examples that oligonucleotide binding is affected bythe formation of an occlusive structure in the target DNA. In each ofthese cases, the oligonucleotides used to bind and capture the targetnucleic acid were designed to be substantially complementary to a singleregion of the target. The following two Examples demonstrate the use ofoligonucleotides that are designed to interact with multiple,non-contiguous regions of the target DNA. In some embodiments of themethods of the present invention, the oligonucleotides (i.e., bridgingoligonucleotides) are designed to interact with regions that are broughtinto close proximity by the formation of folded structure in the targetstrand. By using short sections of complementarity on either side of theconnecting segment, it is intended that the bridge oligonucleotides bedependent on the binding of both of the sections of complementarity, andthat changes in, or the absence of, the intervening folded structurecause a significant change in the affinity between the bridgeoligonucleotide and the target DNA.

EXAMPLE 6 Size of Complementary Regions Affects the Ability of BridgingOligonucleotides to Discriminate Between Targets That Contain IdenticalRegions of Complementarity, But Different Folded Structures

In this Example, the effect of length of complementarity on each side ofthe bridge oligonucleotides on the ability of the bridge oligonucleotideto distinguish between test molecule #80, 81 and 82 (SEQ ID NOS:39-41)was examined. As noted above, these oligonucleotides have identicalregions of complementarity to which the bridge oligonucleotides of thisExample may hybridize. The bridge oligonucleotides used in this test areshown in the lower half of FIG. 11A, arranged in the orientation inwhich they would hybridize to test molecule #80 (SEQ ID NO:39). Threebridging oligonucleotides, shown as #78, #4 and #79 (SEQ ID NOS:42, 43,44), were used, and these had 6, 7 or 8 nucleotides of complementarity,respectively, to each side of the hairpin formed in target #80 (SEQ IDNO:39). The two regions of target complementarity were separated by apair of thymidine nucleotides in each oligonucleotides to provideadditional flexibility to the three-leg junction (Zhong et al.,Biochem., 32:6898 [1993]; and Yang et al., Biochem., 35:7959 [1996]).All the biotinylated oligonucleotides were gel-purified after synthesisusing the standard oligonucleotide purification methods.

In these hybridization analyses, the capture probes were bound to thetarget DNAs in solution and then immobilized on a solid support, asdescribed in the previous Examples. For each of these tests (each of thethree bridge oligonucleotides listed above was tested on each of thethree test molecules), a 150 μl hybridization mixture was assembledcontaining 20 fmols of a fluorescein-labeled test molecule as depictedin FIG. 10 (SEQ ID NOS:39-41), 1.5 pmole of one of the biotinylatedcapture probe 78, 4 or 79 (SEQ ID NOS:42-44), 10 mg/ml tRNA and 0.2%acetylated BSA, in 150 ml of 4.5× SSPE. The mixture was incubated atroom temperature for 30 min.

Aliquots (100 ul) of the mixtures were then transferred to wells in astreptavidin-coated 96-well plate (Boehringer Mannheim) and incubated atroom temperature for 20 min. The plate was then washed three times withTBS (25 mM Tris-Cl, 0.15 M NaCl, pH 7.2) with 0.01% Tween®-20 non-ionicdetergent. Then, 100 μl of a 1:5000 dilution of 0.75 u/mlanti-fluorescein antibody conjugated with alkaline-phosphatase in 0.2%I-block buffer (Tropix, Bedford, Mass.) was added to each well. After 20min at room temperature, the plate was washed three times with TBS with0.01% Tween®-20. Then, 100 μl of Attophos fluorescent substrate (JBL,San Louis Obisbo, Calif.) were added to each well and the plate wasincubated at 37° C. for 1 hour, before fluorescence readings were takenusing a Perkin-Elmer Cytofluor-4000 set to excite at 450/50 mm and toand detect emission at 580/50 nm. Each assay was performed in duplicateand the standard deviation is represented by the black bar at the top ofeach column in the right panel of FIG. 12. In this Figure, thefluorescence intensity is indicated in arbitrary fluorescence units.

The results, shown in FIG. 12, indicate that the bridgingoligonucleotide #79 (SEQ ID NO:44), having 8 bases pairing to each sideof the hairpin in the DNA target, gives better binding activity to thetarget DNA than oligonucleotides that have 7 bases pairing (#4; SEQ IDNO:43), which is better than oligonucleotides that have only 6 basespairing (#78; SEQ ID NO:42). Furthermore, the oligonucleotides with theshorter flanking sequences did not show any significant difference inbinding to the different test molecules, indicating that the presence orabsence of structure was immaterial to their binding under these testconditions. In contrast, the oligonucleotide with the 8 bp flanks had a6 to 7-fold higher affinity for the folded molecules #80 (SEQ ID NO:39)and #81 (SEQ ID NO:40), when compared to the unstructured #82 (SEQ IDNO:41) molecule.

This demonstrated that bridge oligonucleotides are suitable for theassessment of differences in folded structure of a target molecule, incontrast to previous reports (Francois et al., Nucl. Acid. Res. 22: 3943[1994]).

While the 8-bp flanks are clearly the preferred size in thisexperimental system, the absolute number of basepairs required for anyparticular bridge oligonucleotide system may vary other factorsaffecting the stability of the interaction, as discussed above, such aswith the G-C content of the hybridization site, the temperature andsolution conditions under which the reaction is performed, and thenature of the structure to be bridged. Thus, it is contemplated that insome systems, bridge oligonucleotides comprise any appropriate lengthsuitable for the assay system.

EXAMPLE 7 Bridging Oligonucleotides

In this Example, two schemes were investigated in order to determine howthe bridging oligonucleotide might bind to the targeted hairpinstructure, as illustrated in FIG. 11B. Although an understanding of themechanism is not necessary in order to make and use the presentinvention, nor is it intended that the present invention be limited toany particular mechanism, one possibility is that one bridgingoligonucleotide molecule binds to one DNA target molecule, as diagrammedin the top half of the Figure. A second possibility is that two or moreof the bridging oligonucleotide molecules bind to one DNA targetmolecule, with the apparent increase in signal resulting from thepresence of two biotin moieties on the complex facilitating binding ordetection, rather than successfully spanning of a structure by a singlebridge oligonucleotide.

To differentiate these two possibilities, two additionaloligonucleotides were synthesized (oligonucleotide #114 and #115 [SEQ IDNOS:45 and 46, respectively]), as shown in FIG. 11B. Oligonucleotide#114 (SEQ ID NO:45) is almost identical to #79 (SEQ ID NO:44), exceptthat two mutations have been introduced in such way that it cannothybridize to the right side of the hairpin on the target DNA. Similarly,oligonucleotide #115 (SEQ ID NO:46) is a version of #79 (SEQ ID NO:44)having two base mutations so that it can't hybridize to the left side ifthe hairpin on the target DNA. If the ability of oligonucleotide #79(SEQ ID NO:44) to bind to the folded molecules is truly dependent on asingle oligonucleotide bridging the structure then neither of the‘pseudo’ bridge oligonucleotides, #114 or #115 (SEQ ID NOS:45 and 46,respectively), should be able to perform in this way. However, if theincreased binding is in fact due to the presence of two copies of # 79(SEQ ID NO:44), which would be arranged as depicted for #114 and #115(SEQ ID NOS:45 and 46, respectively) in the bottom half of FIG. 11B,then #114 and #115 (SEQ ID NOS:45 and 46, respectively) used togethershould give the same result.

In addition to the test of the bridging function, the necessity of thespacing thymidines in the center of each bridge oligonucleotide wasassessed. An oligonucleotide having the same complementary flankingsequences as oligonucleotide #79, but lacking the two T's in the middle,was created. This oligonucleotide (#116 [SEQ ID NO:47]), is depicted inthe bottom half of FIG. 11A. In addition, to test the necessity ofhaving a physical linkage between the binding halves of #79 (SEQ IDNO:44), to half molecules were created, each having complementarity toone of side of the test molecules, #117 (SEQ ID NO:48) to the right sideand #118 (SEQ ID NO:49) to left side, as depicted in FIG. 11A, and eachhaving one of the two spacer T residues. Finally, two 10-meroligonucleotides were created, each with sufficient contiguouscomplementarity to bind without any bridging activity. One of these wascomplementary to the left flank (#FD91; SEQ ID NO:50), which isunstructured in all cases, while the other was complementary to thesequence involved in the structures of the folded test molecules (#2;SEQ ID NO:51). These are depicted in the top half of FIG. 11A.

The hybridization analyses were performed as described in Example 6,except that 15 fmoles of the fluorescein labeled test molecules wereused, and the amount of bridge oligonucleotide was held to a total of1.5 pmole when #114 and #115 (SEQ ID NOS:45 and 46, respectively) wereused in combination. The results are shown in FIGS. 13A and 13B.

Taking the results in reverse order: the 10-mer control oligonucleotidesshowed the expected profiles in binding i.e., the oligonucleotidecomplementary to the unstructured region, #FD91 (SEQ ID NO:50), boundwith nearly equal affinity to each of the test molecules, while theoligonucleotide complementary to the portion that forms structure inmolecules #80 and #81 (SEQ ID NOS:39 and 40, respectively) bound wellonly to unstructured test molecule #82 (SEQ ID NO:41). This furtherillustrates that structure alone is an important determinant in thebinding of the capture probes in embodiments of the methods of thepresent invention.

When the oligonucleotide without any spacer residues, #116 (SEQ IDNO:47), was tested for its ability to bind the test molecules, it wasfound that this oligonucleotide could not distinguish between the foldedand unfolded molecules (See, FIG. 13A). This demonstrated thathybridization across structures is greatly enhanced by the presence ofsome spacing material between the segments of complementarity.

Finally, the results of testing the pseudo bridge oligonucleotides,separately and in combination, are shown in FIG. 13B. It can be seen bythese data, that oligonucleotides #114 and #115 (SEQ ID NOS:45 and 46,respectively) are not capable, either alone or in combination, toduplicate the binding profile of the true bridge, #79 (SEQ ID NO:44).The enhanced binding to the unstructured test molecule #82 (SEQ IDNO:41) is possibly attributable to the accessibility of this moleculefor binding both oligonucleotides. Note that the fluorescence signalseen with the combination of #s 114, 115 and molecule #82 (SEQ ID NOS:45, 46, and 41, respectively), about 650 fluorescence units, is nearlyidentical to the signal seen when #79 (SEQ ID NO:44) is combined with#82 (SEQ ID NO:41). This supports the idea that two copies of #79 (SEQID NO:44) may be involved in creating the signal with # 82 (SEQ IDNO:41).

It is clear from the above that the present invention provides methodsfor the analysis of the characteristic conformations of nucleic acidswithout the need for either electrophoretic separation of conformationsor fragments or for elaborate and expensive methods of visualizing gels(e.g., darkroom supplies, blotting equipment or fluorescence imagers).The novel methods of the present invention allow the rapididentification of variants (e.g., mutations) within human genes as wellas the detection and identification of pathogens in clinical samples.

The previous examples demonstrated the use of bridging oligonucleotidesto capture specific target molecules through hybridization tonon-contiguous complementary sequences. However, the use of bridgingoligonucleotides is not limited to hybrid capture. Bridgingoligonucleotides hybridizing to folded target molecules can be used inplace of standard oligonucleotides in almost any application, includingapplications in which enzymes modify probes that have found their targetcomplement. Such enzymatic modifications include, but are not limited toprimer extension, ligation and structure-specific nuclease cleavage. Itwill easily be appreciated by those skilled in the art that performanceof bridging oligonucleotides in these basic enzymatic reactions isindicative of their utility in assays that are based on reiterativeperformance of these reactions, including but not limited to cyclesequencing, polymerase chain reaction, ligase chain reaction, cyclingprobe reaction and the Invader™ invasive cleavage reaction. The examplesbelow demonstrate the use of bridging oligonucleotides in each of thebasic enzymatic reaction systems.

EXAMPLE 8 Analysis of Folded Structures of a Hepatitis C Virus-DerivedAmplicon and Design of Bridging Oligonucleotides

The process of identifying candidate structures for bridging with probesinvolves i) pinpointing all modification or cleavage sites; ii)predicting a set of most probable structures, and selecting those thatfit with the specificity of the modification means; and iii) designingand testing probes to span the most probably structures. If desired, theinformation deduced at step ii) can be confirmed by deletion analysissuch as PCR walking, or any equivalent method that allows the selectiverepression or removal of one half of a suspected basepair frominteraction.

This stepwise approach is illustrated here for a 244 nt amplicon derivedfrom HCV type 1a. The identification of the cleavage sites in all fourtypes of HCV amplicon is described in Example 3. FIG. 15 shows sequenceof 5′ UTR region of HCV genotypes 1a (SEQ ID NO:124), 1b (SEQ IDNO:125), 2a/c (SEQ ID NO:126) and 3a (SEQ ID NO: 127) with markedcleavage sites. Note that the designations 2a and 2a/c are usedinterchangeably throughout, and refer to the same HCV viral type, theamplicon of which is SEQ ID NO:22.

The type 1a sequence as then subjected to folding predictions using themfold version 2.3 program, which is available either through GeneticsComputer Group (Madison, Wis.) or through public access to the authors'web site (http://www.ibc.wustl.edu/˜zuker). Folding was done with usingeither DNA or RNA parameters with a selected folding temperature of 37°C. The output was set to include the optimal structure (lowest freeenergy) and any structure with a 20 percent or lower increase incalculated free energy (termed a “suboptimality of 20%”). All otherprogram parameters used the default values. Folding with the RNAparameters generated 32 possible structures, while the DNA parametersgave 18 structures. Two of the structures predicted with the RNAparameters showed the best agreement with the cleavage data from theCFLP® analysis. These structures, the first and the thirtieth out of 32,are depicted in FIGS. 16A (SEQ ID NO:128) and 16B (SEQ ID NO:128).

Structures predicted by the above analysis can be confirmed through theuse of CFLP® analysis on fragments that delete the putative downstreampairing partner (Brow et al., supra). This approach, termed PCR walking,is illustrated here by the confirmation of the pairing partnerresponsible for the CFLP® cleavage at position 161 in the HCV type 1a244 nt amplicon. The mfold program predicted a structure that paired a Gat 161 with a C at position 205 (FIG. 17A, left conformer). To confirmthis two deletion amplicons were made. Each amplicon was 205 nt long.One included the C205 at the 3′ end, while the other substituted a T at205 to disrupt the basepair. PCR was conducted as described in Example3, except the downstream primers 67 and 68 were substituted for (SEQ IDNO:25) used to amplify the full length amplicons. The resulting DNAswere purified and subjected to CFLP® analysis, resolved and visualizedas described in Example 3. The resulting image is shown in FIG. 17B (SEQID NO:128). The identity of residue 205 in the deletion fragments isindicated above each lane, and the sizes of selected cleavage bands, asdetermined by comparison to a sequencing ladder in Example 3, areindicated on the right.

Focusing on the band that was the subject of this analysis, at 161 nt,it can be seen that the amplicon having the natural 205C maintained the161 cleavage, while disruption of this base pair in the 205T fragmentcaused a loss of that band, thus supporting the existence of the 161/205interaction. It should be noted that it is possible that the 205 nt basedoes not interact directly with the 161G, and that the C to T changecaused a conformational change elsewhere, which altered the161-containing structure as a secondary effect. While this is lesslikely, the possibility should always be kept in mind when analyzing thedata, especially if unexpected results arise. Not surprisingly, thedeletions and mutations also give rise to pattern changes elsewhere inthe pattern, indicating how little change is required to be detectableby CFLP®.

Based on the combined CFLP®, nfold, and PCR walking data, three of themost likely conformations for this region were chosen and three bridgeoligonucleotides were designed to span the structures. These are shownschematically in FIG. 17C. The “b” (SEQ ID NO:53) and “n” (SEQ ID NO:65)variants address essentially the same conformation with a differencerelated to the small central stem. Though predicted by mfold, thepresence of this structure is not predicted by the CFLP® pattern for the244-mer (FIG. 17A, right lane). Consequently, bridge probes weredesigned that either spanned that structure (“n”; SEQ ID NO:65) or thatcomplemented the 8 contiguous bases upstream of the larger stem (“b”;SEQ ID NO:53). The “m” (SEQ ID NO:64) bridge probe was designed to crossthe base of the single stem of the other conformer. Each of the theseprobes was tested for binding to the HCV 1a amplicon as described inExample 6. While the “m” (SEQ ID NO:64) and “n” (SEQ ID NO:65) probesfailed to capture significant amounts of target, the “b” (SEQ ID NO:53)probe was found to be effective, as will be illustrated in the followingexamples.

Using the “b” oligonucleotide (SEQ ID NO:53) as a model, a number ofvariant bridges were designed to compare the effects of differentintervening sequences in the probes and on the inclusion of mismatchesin either contact sequence. These bridge probes arc diagrammedschematically as they would align with the HCV 1a predicted structureare shown in FIG. 18A. The connecting line in the center of the “k”probe (SEQ ID NO:56) indicates that the two portions are linked directlytogether without any intervening sequence. Modifications to theintervening region included the use of alternative nucleotides in tolink the contact sequences and the omission of additional interveningnucleotides. A mismatch was included in the middle of either of the twocontact sequences to assess whether the binding of both is necessary forcapture.

The 244 bp target DNAs were created by PCR and isolated as described inExample 3 (SEQ ID NOS:26-29 for types 1a, 1b, 2c and 3a, respectively).The capture probes were synthetically labeled with fluorescein at their5′ end and purified by gel-electrophoresis. The target DNA was labeledwith biotin at the 5′ end of the antisense strand. Each of the theseprobes was tested for binding to the of the HCV amplicons (as shownschematically in FIGS. 18A-18D), as described in Example 6. Each assaywas performed in duplicate and the standard deviation is represented bythe black bar at the top of each column in FIG. 19. The fluorescenceintensity is indicated in arbitrary fluorescence units, shown on theleft side of each chart panel. The probe included in each capturereaction are indicated below each graph column. A control probe notshown in the schematic diagram (49-3; 5° Fl-GCGAAAGGCCTTGTGG; SEQ IDNO:66) that hybridizes to all HCV variants was used with each target toverify the presence and amount of DNA in each reaction. The rightmostcolumn in each panel shows the signal from the control reaction.

These data show that functional bridge oligonucleotides may be designedwith different intervening sequences, or without any interveningsequence at all (“k”; SEQ ID NO:56), although those having extranucleotides showed greater signal in most tests. The low signal seenwhen a mismatch is included on either side verifies that both contactsequences participate in the binding. It is interesting to note that thesignal from oligonucleotide “i” (SEQ ID NO:54) is greater than “b” (SEQID NO:53) in the type 2a/c test. Examination of this junction in FIG.18C shows that this type has a C to T change relative to the type 1a, aT that may interact with one of the A residues in the interveningsequence of the “i” probe (SEQ ID NO:54), thereby strengthening theinteraction. It can be seen here and in later Examples, that thisbridging design does not interact well with the type 3a amplicon,suggesting that this may not be a favored conformation for thisparticular variant. Nonetheless, these data demonstrate the flexibilityavailable to the user in designing suitable bridging probes.

EXAMPLE 9 Primer Extension of Bridging Oligonucleotides

The folding of the 244 bp DNA copy of a segment of the hepatitis C viralgenome is described above. The bridging oligonucleotides designed tohybridize across the deduced structures were used in a primer extensionreaction to show that the presence of folded structures within thetarget would not prevent extension of the probe by a template-dependentDNA polymerase. The 244 bp target DNAs were created by PCR and isolatedas described in Example 8. The bridging primers (a, b, c, d, and e, SEQID NOS:52, 53, 57, 58, and 59, respectively) are shown in FIG. 20A asthey would be expected to hybridize to a folded structure of the HCVtype 1a amplicon. The oligonucleotide indicated as “a” (SEQ ID NO:52),while it may have some complementarity that suggest it may serve as abridge in some conditions, was designed as a non-bridging primer,intended to fully-hybridize to a non-folded target. This is shownschematically in FIG. 20B.

Each primer extension reaction contained either 50 fmole of the 244 bptarget DNA or 10 ng of human genomic DNA (Novagen #69237-1, Madison,Wis.), 1 pmole of the fluorescein-labeled bridge oligonucleotide, 5units of KlenTaq polymerase (Ab Peptides), and 0.1 mM of each dNTP in 10μl of 1× PCR Buffer containing Mg⁺⁺ (Boehringer Mannheim). The assembledreaction mixtures with all the components were heated to 95° C. for 2minutes, then cooled to the 40° C. for 1 hour. The reactions wereterminated by the addition of 5 μl of 95% formamide with 10 mM EDTA and0.02% Methyl Violet. The samples were then heated at 90° C. for 1minute, and aliquots were resolved by electrophoresis through 10%denaturing polyacrylamide (19:1 cross link) with 7 M urea in a buffer of45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. The gel was visualized using anM.D. Scanner (Molecular Dynamics, Sunnyvale, Calif.). The resultingimage is shown in the panel of FIG. 21.

The target DNAs and the bridging primer/probe used in each reaction areindicated. The product of primer extension is indicated by an arrow onthe left of the panel as a 170 bp band. It can be seen from these datathat the “b” bridging oligonucleotide (SEQ ID NO:53) is able to primesynthesis on the folded HCV target of from all viral types, generatingessentially the same level of signal as the non-bridging “a” primer (SEQID NO:52). Examination of the first (left most) lane, in which humangenomic DNA was used in place of the HCV target shows little or no nonspecific priming, demonstrating the specificity of the primers for theHCV folded sequence. When single base mismatches are introduced oneither side of the bridge (as in “c” and “d” primers; SEQ ID NOS:57 and58, respectively) the signal is dramatically reduced. When only the 3′portion of the bridging primer is provided (“e”; SEQ ID NO:59) theextension is also nearly non-existent. These data demonstrate: a) thatboth complementary portions of these bridging oligonucleotides arerequired for the primers extension, demonstrating that theoligonucleotide is truly bridging; and b) that bridging oligonucleotideswith no more than eight contiguous nucleotides of complementarity insingle region can be used to specifically recognize an HCV viralsequence by use of its folded structure.

Above, the performance of a non-bridging oligonucleotide (i.e., anoligonucleotide that hybridizes to a region of contiguous, complementarybases in the target strand), was compared to the performance of thebridging oligonucleotides to assess the effect of the folded targetstructure on the enzyme activity. However, at elevated temperatures thefolded structures may denature, reducing the binding efficiency of thebridging oligonucleotide relative to the non-bridging oligonucleotide.To demonstrate this effect, primer extension experiments were performedat a range of temperatures selected to decrease the presence of suchstructures as diagrammed in FIG. 22.

For this test, only the bridging, the non bridging and the half primer(“a”, “b” and “e”; SEQ ID NOS:52, 53, and 59) were tested. Each primerextension reaction contained 50 fmole of the 244 bp target DNA, 1 pmoleof the fluorescein-labeled bridge oligonucleotide, 5 units of KlenTaqpolymerase (Ab Peptides) and 0.1 mM of each dNTP in 10 ml of 1× PCRBuffer containing Mg++ (Boehringer Mannheim). Reaction mixtures with allthe components were heated to 95° C. for 2 minutes, then cooled to thevarious extension temperatures for 1 hour. Reactions were performed at40° C., 45° C., 50° C., 55° C. and 60° C. The reactions were terminatedby the addition of 5 ml of 95% formamide with 10 mM EDTA and 0.02%Methyl Violet. The products were heated at 90° C. for 1 minute, andaliquots were resolved by electrophoresis through 10% denaturingpolyacrylamide gel (19:1 cross link) with 7 M urea in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA. The gel was visualized using the M.D.Scanner (Molecular Dynamics, Sunnyvale, Calif.). The resulting image isshown in the panel of FIG. 23. The temperatures (° C.) and the primersused for each reaction are indicated above each lane.

The extended products are indicated by an arrow on the left side of thepanel as a 170 bp band. It can be seen from these data that thenon-bridging oligonucleotide (“a”; SEQ ID NO:52) can prime synthesis ateach of the test temperatures. The bridging oligonucleotide (“b”; SEQ IDNO:53), however, loses its ability to prime synthesis as the temperatureof the reaction rises. This further demonstrates that the bridgingoligonucleotides require the presence of the fold within the targetstrand. This also shows that the use of target folded structure toeither support bridging oligonucleotide binding, or to allowstructure-based discrimination of sequences as described in previousexamples, is preferably done at lower temperature that those used fornon-bridging applications. The precise temperature required to maintaina given structure will vary widely depending on the size and stabilityof a given structure, but a simple temperature titration such as isshown here will serve to identify optimal reaction conditions.

It will be appreciated by those skilled in the art that the targetdependent extension of a bridging oligonucleotide can be adapted to thepolymerase chain reaction method of target sequence amplification, usingstandard methods with minimal adaptation. In a PCR, either or both ofthe primers may be selected to perform the initial target recognitionthrough the specific recognition of non-contiguous sequences. Aschematic representation of a reaction in which both primers are thusconfigured in shown in FIG. 34. This is a simplified version of a PCRdiagram that does not show all products at each step; the products shownare selected to demonstrate the manner in which a pair of bridgingoligonucleotides may be designed. This example as described is intendedas an illustrative example and not as a limitation on the mechanisms ofapplication of the present invention. As shown in 34 a, the first strandwould be copied from a folded target strand as described above. Thebridging oligonucleotide would anneal to the target at low temperature(relative to the temperature at which strand extension takes place). Asthe temperature of the reaction increases toward a chosen extensiontemperature (FIG. 34b), the folded structures would be disrupted, butthe now partially extended primer would not disassociate due to itsincreased length. This would allow the polymerase to fully extend theprimer, creating a double strand (FIG. 34c). In the next PCR cycle,after the strands have been denatured by heating, and the reaction hasagain cooled to an appropriate annealing temperature, the newlysynthesized strand would likewise assume distinct folded structures,which can serve as binding sites for a second bridging primer (FIG.34d). When the second primer is fully extended it would fill in theoriginal bridging oligonucleotide with perfectly complementary sequence.In subsequent cycles of the PCR, the former bridge oligonucleotideswould now operate as standard, fully complementary oligonucleotides,amplifying the target region between the 3′ ends of the original bindingsites. The resulting flanking sequences added by the bridgeoligonucleotides would be unique to the bridge sequences.

The selection of conditions for using bridging primers in PCR is notdissimilar in reactions designed to use mismatched or degenerateoligonucleotides (Compton, in PCR Protocols, Innis et al. (Eds.),[1990], at p. 39). In the first few cycles of PCR it would be desirableto use an annealing temperature that would be permissive of the bridgecontact formation. This reaction temperature could be determinedempirically for any bridge oligonucleotide by a number of methods knownin the art, including direct measurement (e.g., in a temperaturecontrolled spectrophotometer), or by the use of the methods presentedhere, such as by plate capture, described in numerous examples above, orby temperature titration, as described in this Example. The principlesof oligonucleotide design for maximum specificity are also similar tostandard practices known in the art. For example, for maximumspecificity of PCR oligonucleotides, it is a common practice to skew thestability such that the 5′ end of the oligonucleotides has a higherlocal stability and the 3′ end has a lower local stability. Conditions(e.g., sufficiently high annealing temperature), are then selected sothat the 3′ terminal sequence is unlikely to successfully bind unlessthe 5′ end also binds. This prevents mis-priming caused by unintendedhybridization of the 3′ terminal residues at non-target sites.

The bridge oligonucleotides can be designed with a similar skew. Inaddition, it is contemplated that the bridge oligonucleotides beselected such that the 340 end is less stable (e.g., through the use ofA/T base pairs or a short contact sequence) so that it is unlikely tofind its target site without the successful binding of the other contactsequences, thereby increasing the discriminating power of the brideoligonucleotides in a PCR assay.

EXAMPLE 10 Hybridization Analysis of the Bridge Oligonucleotide inCombination with a Flanking Oligonucleotide

Several reactions using involving standard probes require hybridizationof two or more oligonucleotides in close proximity. For example, aligation reactions to join oligonucleotide probes requires that at leasttwo probes hybridize adjacently (i.e., without a gap), on a target ortemplate strand. The Invader™ reaction requires oligonucleotides tohybridize either adjacently, or with one or more nucleotides of overlap.In both of these scenarios, the binding of adjacent sites on acomplementary strand means that resulting individual duplex regions arecooperatively stabilized by the coaxial stacking of the helices. Inother words, each duplex will be more stable, i.e., will have a higherapparent melting temperature, in the presence of the other than it wouldin isolation. In the hybridization-based discrimination of genotypesbased on the stability of folded target structure, the increasedstability of binding of the bridge probe may reduce the ability todiscriminate, absent compensating changes in the design of the probe.

To examine the effect of a neighboring oligonucleotide, hybridizationcapture tests were used on the bridging oligonucleotides and neighboroligonucleotides designed for the ligation assay. The oligonucleotideswere tested either alone, or in the pairs as they would be used in theenzymatic assays. For these tests the capture probes (SEQ ID NOS:52, 53,60, and 66) were synthetically labeled with fluorescein at their 5′ endand purified by gel electrophoresis. These probes are among those shownschematically in FIG. 24, identified by lower case letter. The HCVtarget DNA was amplified by PCR as described in Example 3, but the 5′end of the antisense strand was labeled with biotin, instead offluorescein. The primers employed for the amplification of HCV targetDNAs were: 5′ primer: 5′-B-CTCGCAAGCACCCTATCA (SEQ ID NO:24)-and 3′primer: 5′-GCAGAAAGCGTCTAGCCATGG (SEQ ID NO:25). The PCR reactions wereperformed as described in Example 3, and the resulting 244 bp PCRproducts (SEQ ID NOS:20-23) for types 1a, 1b, 2c and 3a, respectively)were purified using “High Pure PCR Product Purification Kit” (BoehringerMannheim) and eluted in dH₂O according to the manufacturer'sinstructions. The same amount of DNA was used for each sample in thecapture assay.

The hybridization analyses were similar to these described in previousexamples. For each test, a hybridization mixture was assembledcontaining 20 fmoles of heat-denatured, 244 bp HCV PCR product, 1 pmoleeach of the fluorescein-labeled bridge oligonucleotides and the ligationoligonucleotide probe depicted in FIG. 24 (“b,” “a,” and “f”. SEQ IDNO:53, 52, and 62, respectively), and 0.01 mg/ml tRNA, in 100 μl of asolution of 0.2% acetylated BSA, 4.5× SSPE. After incubating the mixtureat room temperature for 30 min., the mixtures were transferred intowells of a streptavidin-coated 96-well plate (Boehringer Mannheim) andincubated at room temperature for 30 min. The plate was then washedthree times with 1× PBS, with 0.01% Tween®-20 non-ionic detergent,containing 0.2% I-Block (Tropix, Bedford, Mass.). A 1:5000 dilution of0.75 u/ml anti-fluorescein antibody conjugated with alkaline-phosphatasein 0.2% I-block buffer was added to each well. After 20 min at roomtemperature, the plate was washed three times with TBS (25 mM Tris-Cl,0.15 M NaCl, pH 7.2). One hundred microliters of Attophos® fluorescentsubstrate (JBL) was added to each well and the plate was incubated atroom temperature for 1 hour before fluorescence readings were takenusing a Perkin-Elmer Cytofluor-4000 set to excite at 450/50 nm and toand detect emission at 580/50 nm. Each assay was performed in duplicate,and the standard deviation is represented by the black bar at the top ofeach column in FIG. 25. In this Figure, the fluorescence intensity isindicated in arbitrary fluorescence units, shown on the left side ofeach chart panel. The probes included in each capture reaction areindicated below each graph column. A control probe not shown in theschematic diagram (“49-3”; 5° Fl-GCGAAAGGCCTTGTGG; SEQ ID NO:66) thathybridizes to all HCV variants was used with each target to verify thepresence and amount of DNA in each reaction. The leftmost column in eachpanel shows the signal from the control reaction.

In addition, a comparison of bridging and non-bridging oligonucleotidesfor HCV capture was conducted. It can be seen by comparing the signalsfrom the “a” (non-bridging) and “b” probes (SEQ ID NO:52 and 53,respectively), that the bridge oligonucleotide, having only 8 nts ofuninterrupted complementarity to the target, binds to the targets withnearly the same affinity as the 18 nt, fully complementaryoligonucleotide, demonstrating the efficacy of the bridge design. Eachof the oligonucleotides binds most strongly to HCV type 1a, slightlyless efficiently to types 1b and 2a/c , and not very strongly to type3a. The degree to which this differential binding is out of proportionto variations seen with the control oligonucleotide, particularlyevident with type 3a, further illustrated the ability of these probes todifferentiate types based on folding of the target nucleic acid.

Effect of a neighboring oligonucleotide on the bridge binding signal.The Probe “g” (SEQ ID NO:60), a probe used in an Invader™ cleavage assayand diagrammed in FIG. 29, was included because it has the sametarget-complementary sequence as the “f” probe (SEQ ID NO:62), but italso has a 5′ fluorescein label to allow it to serve as a capture probe,whereas “f” does not, because it is intended for ligation. The “g” probe(SEQ ID NO:60) also comprises a short 540 tail of 4 T residues that arenot included in “f” (SEQ ID NO:62). While not identical in composition,the capture signal from “g” (SEQ ID NO:60) should be a good indicator ofthe strength of the interaction between the HCV targets and the “f” (SEQID NO:62) oligonucleotide. The base signal from each of the captureoligonucleotides (columns marked underneath as “b” and “a”), and theeffect of the addition of a neighboring oligonucleotide can be seen byexamining the signal in reactions that included the ligation probe “f”(SEQ ID NO:62). It can be seen by comparing “a” to “a/f′ that thepresence of the second oligonucleotide has little or no effect on thecapture of these HCV targets with the non-bridging “a” probe (SEQ IDNO:52). In contrast, in all cases the addition of the “f”oligonucleotide (SEQ ID NO:62) substantially increases the binding bythe bridging “b” (SEQ ID NO:53) oligonucleotide. Because “f” (SEQ IDNO:62) is unlabeled and does not contribute to either the plate bindingor the signal generation, the additional signal seen in these columnsmust come from increased binding of “b” (SEQ ID NO:53). This increasedstability of binding using a flanking oligonucleotide may be used toenhance the performance of the bridge oligonucleotides in capturing alltypes of a target. Conversely, the increased stability must beconsidered in the design of the bridge oligonucleotides only if the goalis to create a system that is maximally sensitive to subtle structuralchanges, as described in Example 7. When maximum discrimination isdesired in an assay that requires the binding of an adjacentoligonucleotide, it may be desirable to shorten or otherwise reduce thestability of the contact segment of the bridge that is nearest to theneighboring oligonucleotide. Common methods of reducing oligonucleotidebinding affinity, such as through the use of base analogs or mismatchesare well known in the art.

EXAMPLE 11 Target Dependent Ligation of a Bridging Oligonucleotide to anAdjacent Oligonucleotide.

To examine the mismatch effect on the ligation between a bridgingoligonucleotide and the ligation oligonucleotides, a linear (i.e.,non-folded) oligonucleotide target having appropriately oriented regionsof complementarity was synthesized for use as a control target (SEQ IDNO:63)(i.e., to examine the effect of ligation in the presence of astem). This control target aligned with the ligation and bridgingoligonucleotides is depicted in FIG. 26. The PCR conditions to prepare244 bp ds HCV target DNA were the same as described above.

Each ligation reaction contained 200 fmole of the target DNA, 1 pmoleeach of the bridging and ligation oligonucleotides, 100 units ofAmpli-ligase® (Epicenter) in 10 μl of 1× Ampli-ligase® buffer(Epicenter). A control reaction was performed without target DNA.Reactions were assembled with all components except the enzyme and theenzyme buffer, heated to 95° C. for 3 minutes, then cooled to thereaction temperature of 45° C. The ligation reactions were started withthe addition of the enzyme and the enzyme buffer, and incubated for 1hour. The reactions were terminated by the addition of 4 μl of 95%formamide with 10 mM EDTA and 0.02% Methyl Violet. The products wereheated at 90° C. for 1 minute, and aliquots were resolved byelectrophoresis through 15% denaturing polyacrylamide gel (19:1 crosslink) with 7 M urea in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mMEDTA. The gel was visualized using the M.D. Scanner (Molecular Dynamics,Sunnyvale, Calif.). The resulting image is shown in the panel of FIG.27. The sizes in nucleotides of each band is indicated on the left sideof the panel.

The labeled, unreacted probes are visible as either an 18 nt band (a-d:i.e., probes corresponding to SEQ ID NOS:52, 53, 57, and 58) or an 8 ntband (e; i.e., probe corresponding to SEQ ID NO:59). The product ofligation between oligonucleotide “f” (SEQ ID NO:62) and bridge probes“a” through “c” (SEQ ID NOS:52, 53, and 57, respectively), is visible asa 33 nt band near the top of the panel, while the product of ligationbetween “f” (SEQ ID NO:62) and “e” (SEQ ID NO:59) is indicated as a 23nt band. It can be seen from these data that all of the bridgeoligonucleotides are able to use the folded target at a template tocorrectly align for ligation. The efficiency of the ligation can beassessed by comparing the product intensity in each lane to theintensity from ligation of the non-bridging oligonucleotide “a” (SEQ IDNO:52). Probe “b” (SEQ ID NO:53), which is fully complementary in bothcontact sequences shows the strongest signal on the HCV type 1a, whichis consistent with the binding seen in the capture tests of theseoligonucleotides. The ligation of the shortest oligonucleotide, “e” (SEQID NO:59) shows that even an 8 nt probe is sufficiently stable in thisassay to be ligated at some level. The least amount of ligation is seenwith the bridge probe having the mismatch closest to the site ofligation, reflecting a decrease in hybridization for this portion of theoligonucleotide or a decrease in activity of the ligase enzyme near amismatch, or a combination of these effects.

As described above for the primer extension of the bridgingoligonucleotide, at elevated ligation temperatures the folded structuresdenature, reducing the binding efficiency of the bridgingoligonucleotide relative to the non-bridging oligonucleotide. To examinethis effect in a ligation reaction, and to examine the effect of thefolding on the discrimination of the amplicons by HCV type, additionalexperiments were performed on all four amplicon types, at a range oftemperatures. Because the thermostable ligase activity intended for useunder high-stringency conditions (e.g., at temperatures above about 45°C.), T4 DNA ligase, commonly used at 10 to 30° C., was used in theligations performed at lower temperature.

Each ligation reaction contained 200 fmole of the target DNA, 1 pmole ofthe fluorescein-labeled bridge oligonucleotide, 1 pmole of the ligationoligonucleotide and 3 units of T4 Ligase (Promega) in 10 μl of 1× T4Ligase® buffer (Promega). Reactions were assembled with all componentsexcept the enzyme and the concentrated enzyme buffer, heated to 95° C.for 3 minutes, then cooled to the reaction temperature of either 25° C.or 45° C. The ligation reactions were started by the addition of theenzyme and the concentrated buffer to bring each of those components tothe final concentrations listed above, and incubated for 1 hour. Thereactions were terminated by the addition of 4 μl of 95% formamide with10 mM EDTA and 0.02% Methyl Violet. The products were heated at 90° C.for 1 minute, and aliquots were resolved by electrophoresis through 15%denaturing polyacrylamide (19:1 cross link) with 7 M urea in a buffer of45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. The gel was visualized using theM.D. Scanner (Molecular Dynamics, Sunnyvale, Calif.). The resultingimage is shown in the panel of FIG. 28. The reaction temperatures areindicated at the top of the panel, and the control reactions lacking theligase enzyme are indicated. The labeled, unreacted probes are visibleas an 18 nt band. The product of ligation is visible as a 33 nt bandnear the top of the panel.

Examination of the product bands at the two temperatures confirms theexpected increase in discrimination at the lower temperature. Thesignals from the 1a and 1b types are very similar, while the signalsfrom 2a/c and 3a are much lower. While the 3a result is consistent withthe capture data using the combination of the “b” and “f” probes (SEQ IDNO:53 and 62, respectively) shown in FIG. 25, the signal from 2a/c isrelatively lower than in the capture. Without limitation to anyparticular mechanism, this effect may be attributable to the substratespecificity of the ligase at this temperature (e.g., the assumedstructure may have a loop or bulge situated in a manner that inhibitsthe enzyme). Nonetheless, this example demonstrates that these viraltypes may be distinguished using ligation reactions performed undernon-stringent conditions. At slightly elevated temperature, the productbands are of approximately equal, and stronger intensity. The uniformityof the signal may be attributed to the partial or complete disruption ofthe structure at this temperature. It was observed in the FIG. 27 thateven the 8 nt “e” (SEQ ID NO:59) control molecule could be efficientlyligated to the “f” ligation oligonucleotide (SEQ ID NO:62) on the linearsynthetic target (“S.T.”; SEQ ID NO:63). This indicates that the ligasecan join rather short oligonucleotides, even at temperatures above theirestimated Tm. As the structure is unfolded in the 45° C. reaction inFIG. 28, the bridging oligonucleotide may be participating in theligation in this manner (i.e., only its 3′ end is binding), eliminatingthe ability to discriminate between types under these conditions. Thestrength of the signal may reflect increased activity of the enzyme atthis temperature, the preference for the enzyme for this structure overthe bridge conformation, or a combination of these or other factors.

The ligation under the lower temperature conditions demonstrates thatbridging oligonucleotides can be used to identify folded targetmolecules in this type of a reaction. Since the contact sequence on the3′ terminus of the bridging oligonucleotides of these examples isclearly stabilized in these reactions (i.e., a mismatch in this portion,as in oligonucleotide “c” (SEQ ID NO:57), has less effect on the bridgeactivity of the probe than in the capture, primer extension and cleavageassays shown in other examples) it may be desirable to provide a lessstable contact sequence in this region. Means for reducingoligonucleotide Tm are well known in the art, and a few methods arediscussed above, in the context of PCR primer design.

Just as the conditions for bridge oligonucleotide primer extension canbe adapted to the polymerase chain reaction for amplification of signal,the ligation of the bridge oligonucleotides can be adapted to the ligasechain reaction. The target-specific ligation event can be viewed ascreating a unique molecule to be detected, even if the ligation point innot centered, as it is in the LCR. Two possible configurations aredepicted schematically in FIG. 35. In all panels of this Figure, theligation junction is represented by a carat point on the ligated nucleicacid. In the first panel, FIG. 35a, the bridging oligonucleotide wouldbe extended by addition of a short sequence, such as a hexamer or anoctamer. Ligation of short oligonucleotides that are stabilized bycoaxial stacking is known in the art (Kaczorowski and Szybalski, Gene179:189 [1996 ]), and is demonstrated by ligation of the “e”oligonucleotides (SEQ ID NO:59) shown in FIG. 27. The configurationshown in 35 b instead shows the ligation of two longer probes, each ofwhich bridges in a structure. It is contemplated that otherconfigurations within the scope of the present invention would beapparent to those skilled in the art, including but not limited toligation of a non-bridging oligonucleotide to the 5′ end of a bridgingoligonucleotide, or ligation of more that two oligonucleotides assembledon a single folded target.

In each of the embodiments and configurations listed above, the ligationevent would create a unique contiguous sequence not found in the targetnucleic acid. This unique sequence may then itself be detected by anumber of means, including, but not limited to the ligase chainreaction. Practice of the ligase chain reaction for the detection ofspecific sequences is well known in the art, and the means of adaptingthe bridging ligation to this amplification method are easilyascertainable from the literature (See e.g., Barany, PCR Meth. App. 1:5[1991], and U.S. Pat. No. 5,494,810, herein incorporated by reference).The bridging oligonucleotides may also be used in modified LCR assays,such as gap-filling LCR (See e.g., U.S. Patent No. 5,427,930, hereinincorporated by reference), or other variants of the method. Bycombining the bridging oligonucleotides of the present invention withthe ligase chain reaction an investigator can derive the benefits ofstructure characterization discussed above, but performed directly onsamples of interest, without intervening culture or PCR amplification.

EXAMPLE 12 Target Dependent Cleavage of A Probe, Directed by an InvasiveBridging Oligonucleotide

The previous examples demonstrated the ability of the bridgingoligonucleotides to serve as substrate in reactions that produced amaximum of one event for each copy of a folded target. There are manyapplications based on the use of oligonucleotides in which the reactionsare configured to produce many signals for each copy of a target nucleicacid. Such reactions include, but are not limited to ligase chainreaction, polymerase chain reaction, cycle sequencing, and nucleasedetection assays such as the cycling probe reaction. We show here thatsuch reactions can be configured to make use of noncontiguous probebinding. The use of bridging probes may in some embodiments allow thekind of structure-based typing described above to be used in a reactionthat can also amplify the signal from the target. It is also well knownthat even single-stranded nucleic acid targets can fold such that verylittle sequence is actually available for probe binding for detection orfor antisense applications. The ability of probes to bind tonon-contiguous sites facilitates the design of probes that interact onlywith the outer surface of the target nucleic acid, thus allowingdetection or typing of targets that could not previously becharacterized by hybridization methods.

The Invader™ reaction involves the contacting of a target nucleic acidwith a pair of oligonucleotides to create a cleavage structure asdescribed above. The signal probes can leave the structure aftercleavage, to be replaced by an uncleaved copy, thus starting the cycleagain, and allowing each target to create many copies of the cleavedprobe during the course of the reaction. The probes and targets used forthis assay are diagrammed in FIGS. 29A, 29B and 31. The effects of thesignal probe (“g”; SEQ ID NO:60) on the stability of the bridgeoligonucleotides was described in Example 9.

In the experiments in this Example, all invasive cleavage reactionsincluded a mixture of 10 fmole of either the 244 bp target DNA or thesynthetic linear target, 10 pmole each of a fluorescein-labeled bridgeoligonucleotide and the fluorescein-labeled probe (“g” or “h” SEQ ID:60or 61), 10 mM MOPS, 7.5 mM MgCl2, 20 ng of the 5′ nuclease AfuFENI(i.e., a FENI from Archaeoglobus fulgidus, PCT/US97/21783, hereinincorporated herein by reference), and water to a final volume of 10 μl.Reactions were assembled with all components except the enzyme and 7.5mM MgCl₂, heated to 95° C. for 2 minutes. The reactions were then cooledto the indicated reaction temperatures, started with the addition ofenzyme and 7.5 mM MgCl₂, and incubated for 1 hour. The reactions werethen terminated by the addition of 10 μl of 95% formamide with 10 mMEDTA and 0.02% Methyl Violet. The products were heated at 90° C. for 1minute, and aliquots were resolved by electrophoresis through 20%denaturing polyacrylamide gel (19:1 cross link) with 7 M urea in abuffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. The gel was visualizedusing the M.D. Scanner (Molecular Dynamics, Sunnyvale, Calif.).

The first assay tested the ability of both the HCV variants and asynthetic non-folded target to serve as a target in this assay. Allreactions used the “g” signal probe (SEQ ID NO:60), and were incubatedat 55° C. The resulting image is shown in FIG. 30. The type target DNAand the bridging probe used in each assay are identified above eachline. In this Figure, the unreacted probes are indicated with arrows andtheir letters to the left of the panel, in addition, the 4-nt product ofthe cleavage is also indicated by arrow.

Examination of the intensity of the 4 nt band in each lane shows that oneach type of folded target (1a, 1b, 2a/c and 3a) the bridging probe “b”(SEQ ID NO:53) performed nearly as well as the linear probe “a” (SEQ IDNO:52) at directing cleavage of the signal probe “g” (SEQ ID NO:60). Incontrast, the bridging probes either having a mismatch in one contactsequence (“c” and “d”; SEQ ID NOS:57 and 58) or missing one contactsequence (“e”; SEQ ID NO:59) were not able to complete the cleavagestructure to any significant extent. This demonstrates not only that abridging oligonucleotide having no more than 8 bases of contiguouscomplementarity in any contact sequence can nonetheless specificallydetect this HCV sequence, it also shows that both of the contactsequences in the probe are important to this function.

The signal generated from the non-folded synthetic target shown themaximum product yield that can be expected from these probes whenessentially perfectly bound. As expected based on previous experimentsconducted during the development of the present invention, the signal isstronger, although not astoundingly so. Also as expected based onprevious experiments conducted during the development of the presentinvention, the half molecule, which does not cross a structure on thefolded target, does not improve much in performance when the structureis removed, while the non bridging probe performance is decreasedbecause has a number of mismatches to this target (See, FIG. 29B).

As described above for the primer extension and ligation of the bridgingoligonucleotides, at elevated temperatures the folded structuresdenature, reducing the binding efficiency of the bridgingoligonucleotide relative to the non-bridging oligonucleotide. To examinethis effect in an Invaderm reaction, additional experiments wereperformed at a range of temperatures. Because the Invader™ assay isperformed near the Tm of the signal probe to allow turnover withoutthermal cycling, a shorter probe molecule (“h”; SEQ ID NO:61) was madefor use at the lower temperatures. This is shown schematically in FIG.31. The Invader™ reactions were performed as described above, using thebridging probe “b” (SEQ ID NO:53) and the “h” signal probe (SEQ IDNO:61), with incubations done at 30°, 35° and 40 ° C. All four HCVamplicon types were tested. The resulting image is shown in the panel ofFIG. 32. The probes and targets used in each reaction, and thetemperatures of the incubation are indicated above the panel. The arrowon the right indicate the unreacted probes by their letters, and the 4nt cleavage product. The fluorescence, in arbitrary fluorescence units,measured for each of the 4 nt bands is shown below each lane; the samelocation in a no-probe reaction lane was counted to determine thebackground level (in parentheses), which was subtracted from the productcount for each lane.

Examination of these data show that while the “b” (SEQ ID NO:53) bridgefunctions in the invasive cleavage at all temperatures, the lowertemperature reactions show a greater signal differential between the HCVtype 3a lane and the other types. This is consistent with the data fromthe capture experiments described in Examples 8 and 10, showing that the3a type amplicon does not have the same structure in this region as theother 3 types tested. This also demonstrates that discrimination ofsubtle sequence differences by this method is most easily done attemperatures that encourage folding in the target molecules.

This is further supported by examination of the reactions data shown inFIG. 33. This panel compares the signals generated at two temperatures,55° C. and 35° C., using the whole array of bridging and non-bridgingprobes, on a number of targets. The identities of the target DNAs andprobes used in each reaction are indicted above each lane, and thecleavage probes used are indicated below the lanes. The unreacted probesare indicated by arrows and their letters on either side of the panel,and arrows indicate the 4 nucleotide (nt) product of cleavage. Thefluorescence, in arbitrary fluorescence units, measured for each of the4 nt bands is shown below each lane; the same location in a no-probereaction lane was counted to determine the background level (inparentheses), which was subtracted from the product count for each lane.

The data shown in FIG. 33 shows the same profile of detection signal forthe HCV samples as in the previous example, and further demonstratedthat the mismatched bridge probes (“c” and “d”; SEQ ID NO:57 and 58) andthe half probe (“e”; SEQ ID NO:59) have limited function in this assay.Similarly, the probe is not detectably cleaved when the bridgingoligonucleotide is altogether omitted. Furthermore, reactions usinghuman genomic DNA in place of the HCV target exhibit no signal that canbe seen above background, demonstrating the specificity of this assay inboth “stringent” and “non-stringent” conditions.

EXAMPLE 13 Structure Analysis and Bridging Probe Binding to DNA DerivedFrom a Gene Associated With Antibiotic Resistance in Mycobacteriumtuberlosis

In the past decade there has been a tremendous resurgence in theincidence of tuberculosis in this country and throughout the world.Worldwide, the number of new cases reported annually is forecast toincrease from 7.5 million in 1990 to 10.2 million by the year 2000. Analarming feature of this resurgence in tuberculosis is the increasingnumbers of patients presenting with strains of M. tuberculosis that areresistant to one or more anti-tuberculosis drugs (i.e., multi-drugresistant tuberculosis [MDR-TB]).

Resistance to either or both of the antibiotics rifampin (rif) andisoniazid (inh) is the standard by which M. tuberculosis strains arejudged to be multi-drug resistant. Both because of their potentbactericidal activities, and because acquisition of primary resistanceto these drugs is rare (the spontaneous mutation rate of resistance torifampin is approximately 10⁻⁸ and to isoniazid, 10⁻⁸ to 10⁻⁹), untilvery recently, these two antibiotics were among the most powerfulfront-line drugs used to combat the advance and spread of tuberculosis.However surveys of tuberculosis patients in the U.S. reveal that as manyas one-third were infected with strains resistant to one or moreanti-tuberculosis drugs; greater than 25% of the M tuberculosis culturesisolated were resistant to isoniazid and 19% were resistant to bothisoniazid and rifampin (Frieden et al., New Eng. J. Med. 328:521[1993]). Resistance to rifampin is associated with mutation of the rpoBgene in M. tuberculosis. It has been shown that key mutations in thisgene can be detected and identified using the CFLP® method of structureanalysis, demonstrating that these mutations influence the foldedconformations of these genes (Brow et al., J. Clin. Microbiol., 34:3129[1996]; and PCT International Application No. PCT/US95/14673 [WO96/15267]; co-pending application Ser. No. 08/484,956 and 08/520,946).We therefore chose this gene as a model to demonstrate the process ofidentifying non-contiguous sequences that are brought into sufficientlyclose proximity by strand folding for interaction with bridging probes.

The Description of the Invention outlines a step-wise procedure foranalysis of a target secondary structure and for the design of bridgingprobes to interact with any folded nucleic acid molecule. This processcomprises the steps of: a) performing CFLP® analysis to identifynucleotides that are basepaired on the 5′ sides of stems; b) using thispartial basepair information as a “soft constraint” in a fold-predictionprogram such as mfold to produce schematic diagrams (or other suitableoutput) of possible folded conformations that are consistent with theCFLP® data; c) using PCR deletion and directed mutagenesis to confirmthe identities of the nucleotides on the 3′ sides of stems to which the5′ side nucleotides are hydrogen bonded; d) using this full basepairinformation as a “hard constraint” in the fold prediction program toproduce a highly refined set of predicted structures; and e) designingand testing bridging probes that interact with the predicted stems.Depending on the complexity of the data generated at each step, one ormore of steps (a) through (d) may be omitted in any particularapplication. As noted in the Description section, a number of physicalanalytical methods may be combined with a number of secondary structureprediction algorithms to perform this type of analysis; the use CFLP®cleavage method in conjunction with the mfold software is discussed hereas a convenient example and is not presented as a limitation on thescope of the present invention.

To demonstrate the analysis on a non-viral target, DNA fragments wereamplified from the rpoB gene of M tuberculosis. DNA extracted from Mtuberculosis culture was obtained from the CDC (Center for DiseaseControl, Atlanta, Ga.). Genomic DNA was prepared at the CDC usingsiliconized glass beads as described previously (Plikaytis et al., J.Clin. Microbiol. 28:1913 [1990]). A 193-bp fragment of the rpoB gene(SEQ ID NO:69) was generated by PCR amplification of the genomic DNAsample using primers rpo 105 (forward) CGT GGA GGC GAT CAC ACC GCA GACGT (SEQ ID NO:70) and rpo 273 (reverse) GAC CTC CAG CCC GGC ACG CTC ACGT (SEQ ID NO:71). This fragment contains the 81-bp rifampin resistanceregion. This amplicon was cloned using the TOPO-TA cloning kit(K4550-40, Invitrogen, Carlsbad, Calif.). A 128 bp subfragment of therpoB gene (SEQ ID NO:72) was amplified from the resulting plasmid usinga TET-labeled forward primer with the sequence5′-CGCCGCGATCAAGGAGTTCT-3′ (SEQ ID NO:73) and a reverse primer with thesequence 5′-GCTCACGTGACAGACCGCCG-3′ (SEQ ID NO:74). PCR reactions weredone in a final volume of 100 μl, containing: 2 ng of genomic DNA, 35pmoles of each primer, 50 μM of each deoxyribonucleotide (Perkin Elmer,Foster City, Calif.), 1× PCR buffer (20 mM Tris-HCl pH 8.5, 50 mM KCl,1.5 M MgCl₂, 0.05% Tween 20, 0.05% NP40), 1M betaine, 5% DMSO, and 2.5units of Taq polymerase. PCR cycling conditions consisted of an initialdenaturation at 95° C. for 5 minutes, 30 cycles of denaturation at 94°C. for 1 minute, annealing at 58° C. for 1 minute, and extension at 72°C. for 1 minute, with a final 7 minute extension at 72° C. Following PCRamplification, the fragments were purified by treatment with ExonucleaseI (United States biochemical, Cleveland, Ohio) at 37° C. for 45 min, andfollowed with the High Pure PCR Product Purification Kit spin columns(Boehringer Mannheim, Indianapolis, Ind.). The purified products werequantified using the PicoGreen™ assay (Molecular Dynamics, Eugene,Oreg.) according to the manufacturers' recommended procedure. The samePCR procedure was used in the generation of the truncated and mutatedamplicons described below; the forward primer was not varied, and thereverse and mismatch primers were one of the following (the primer namesindicate the construct to be created): 75-121(reverse)TGACAGACCGCCGGGCCC (SEQ ID NO:75) to generate the 121 fragment (SEQ IDNO:76); 75-121(mismatch) AGACAGACCGCCGGGCCC (SEQ ID NO:77) to generatethe 121 mismatch fragment (SEQ ID NO:78); 57-119(reverse)ACAGACCGCCGGGCCCCA (SEQ ID NO:79) to generate the 119 fragment (SEQ IDNO:80); 57-119(mismatch) CCAGACCGCCGGGCCCCA (SEQ ID NO:81) to generatethe 119 mismatch fragment (SEQ ID NO:82); 53-118(reverse)CAGACCGCCGGGCCCCAG (SEQ ID NO:83) to generate the 118 fragment (SEQ IDNO:84); 53-118 (mismatch) GAGACCGCCGGGCCCCAG (SEQ ID NO:85) to generatethe 118 mismatch fragment (SEQ ID NO:86); 62-114(reverse)CCGCCGGGCCCCAGCGCCGA (SEQ ID NO:87) to generate the 114 fragment (SEQ IDNO:88); 62-114(mismatch) GCGCCGGGCCCCAGCGCCGA (SEQ ID NO:89) to generatethe 114 mismatch fragment (SEQ ID NO:90); 63-113(mismatch)CGGCCGGGCCCCAGCGCCGA (SEQ ID NO:91) to generate the 114 mismatch(113)fragment (SEQ ID NO:92); 69-110(reverse) CGGGCCCCAGCGCCGACA (SEQ IDNO:93) to generate the 110 fragment (SEQ ID NO:94); 69-110(mismatch)AGGGCCCCAGCGCCGACA (SEQ ID NO:95) to generate the 110 mismatch fragment(SEQ ID NO:96); 78-106(reverse) CCCCAGCGCCGACAGTCG (SEQ ID NO:97) togenerate the 106 fragment (SEQ ID NO:98); 78-106(mismatch)TCCCAGCGCCGACAGTCG (SEQ ID NO:99) to generate the 106 mismatch fragment(SEQ ID NO:100); 63-87(reverse) CGCTTGTGGGTCAACCCCGA (SEQ ID NO:101) togenerate the 87 fragment (SEQ ID NO:102); and 63-87(mismatch)AGCTTGTGGGTCAACCCCGA (SEQ ID NO:103) to generate the 87 mismatchfragment (SEQ ID NO:104). For all rpoB capture experiments the ampliconswere labeled on the sense strand with biotin instead of TET.

CFLP scanning reactions were performed using 15 ng (175 fmoles) ofpurified PCR product, diluted to a final volume of 15 μl with distilledwater. Optimal CFLP conditions were determined as described previously.Briefly, matrices of three different reaction times (2, 4, and 6minutes) and five temperatures (40, 45, 50, 55, and 60° C.) wereexamined. Conditions were chosen as optimal yielded patterns with anapproximately even distribution of long and short cleavage products. Thediluted amplified fragments were denatured for 15 seconds at 95° C.,cooled to the reaction temperature (50° C.), and combined with 5 μl ofenzyme mixture so that the final 20 μl volume contained: 25U of CleavaseI enzyme, 0.5 mM MnCl₂, 1 mM MgCl₂ and 1× CFLP buffer (10 mM MOPS, pH7.5, 0.05% Tween 20, 0.05% NP40). Reactions were stopped after 4 minutesby the addition of 16 μl of stop buffer (95% formamide with 10 mM EDTA,pH 8.0 and 0.02% methyl violet). The cleavage products were resolved ona 15% denaturing PAGE (19:1 crosslink) containing 7M urea in 0.5× TBE.The resulting pattern was visualized using a Hitachi FMBIO-100fluorescence image analyzer, equipped with a 585 nm filter.

The CFLP® analysis of the 128 nucleotide segment of rpoB identified keybands of 45, 53, 57, 62, 69, 75, 78, and 84 nucleotides in length, amongothers within the CFLP® pattern, as indicated in FIG. 36. These majorband positions were chosen for further analysis. As described above, thespecificity of the Cleavase® I enzyme dictates that these nucleotidesare basepaired to some nucleotide downstream in the strand in thestructure that is cleaved.

Structure analysis of this amplicon using the mfold 2.3 software withoutany added constraints from the CFLP® pattern yielded only seven possiblestructures. Given the small number, manual analysis was sufficient toselect from these 2 variants that together accounted for the majorcleavage products seen in FIG. 36. The cleavage sites are indicated onstructures shown in FIG. 37A (SEQ ID NO:72) (structures generated usedthe hard constraints from PCR walking data, described below).

The structure and cleavage analysis of the structure(s) contributing tothe CFLP® band at position 62 are used here to demonstrate the nextsteps of the process. In both of the structures shown in FIG. 37A (SEQID NO:72), the C at nucleotide 62 is indicated to basepair with a G atnucleotide 114. The stem formed between these positions is the same inboth structures, and is reproduced at the top of FIG. 38A. One step inconfirming the interaction between these bases is to create a truncatedversion of this strand in which nucleotide 114 is changed to preventpairing with nucleotide 62, and examine the resulting CFLP® cleavage(this is termed “PCR walking” in this discussion). This is shownschematically as the variant number 2 (SEQ ID NO:90), the centerstructure at the bottom of FIG. 37B. A control molecule that issimilarly truncated, but that retains the putative 62/114 base pair isshown on the left as variant 1 (SEQ ID NO:88). The CFLP® patterns fromthese 2 molecules are shown in the gel image at the right of FIG. 37B,with an arrow indicating the band at position 62. It can be seen by thedata in the first lane that the CFLPE pattern gives a strong signal atposition 62 in the truncated control, confirming that nucleotide 62 doesnot require any of the material downstream of nt 114 (deleted in thisconstruct) to basepair. Analysis of the variant with the disruptedbasepair in lane 2 shows that removal of the 62/114 basepair shiftscleavage by one position, to the 63/113 basepair. Further variation toremove the 63/113 pairing, by changing nucleotide 113 as diagrammed invariant 3 (SEQ ID NO:92) on the right, nearly eliminates this short stemregion, and eliminates this particular CFLP® band from the patternaltogether (lane 3; the factors contributing to the slight residualsignal at this position will be discussed below). This shows how thecombination of truncation and mutation combined with CFLP® cleavage canbe used to interrogate and confirm specific basepairs within predictedstructures, thereby allowing their use as “hard constraints” in furthercomputer-based modeling. The structures shown in FIG. 37A (SEQ ID NO:72)were generated using the hard constraints determined by such PCRwalking. It is not required that further computer analysis be donebefore bridging probes are designed. If desired, bridge probes may bedesigned on the strength of the PCR walking data.

Based on the data shown in FIG. 37B, several bridging probes weredesigned to span the base of this stem. For all rpoB captureexperiments, the amplicons were labeled on the sense strand with biotininstead of TET. In these capture analyses, the capture probes were boundto the target DNAs in solution and then the complexes were immobilizedon a solid support, as described in Example 8. For each assay, ahybridization mixture was assembled containing 20 fmols of abiotin-labeled test molecule, 1.5 pmole of a fluorescein-labeled captureprobe, 10 μg/ml TRNA, and 0.2% acetylated BSA, in 150 μl of 4.5× SSPE.The mixture was incubated at room temperature for 30 min.

Aliquots (100 μl) of the mixtures were then transferred to wells in astreptavidin-coated 96-well plate (Boehringer Mannheim) and incubated atroom temperature for 20 min. The plate was then washed three times withTBS (25 mM Tris-Cl, 0.15 M NaCl, pH 7.2) with 0.01% Tween®-20 non-ionicdetergent. Then, 100 μl of a 1:5000 dilution of 0.75 u/mlanti-fluorescein antibody conjugated with alkaline-phosphatase in 0.2%I-block buffer (Tropix, Bedford, Mass.) was added to each well. After 20minutes at room temperature, the plate was washed three times with TBSwith 0.01% Tween®-20. Then, 100 μl of Attophos fluorescent substrate(JBL, San Louis Obisbo, Calif.) were added to each well and the platewas incubated at 37° C. for 1 hour, before fluorescence readings weretaken using a Perkin-Elmer Cytofluor-4000 set to excite at 450/50 nm andto and detect emission at 580/50 nm. Each assay was performed induplicate with the standard deviation represented by the black bar atthe top of each column in each graph.

The oligonucleotides designed to bind this stem are shown schematicallyin FIG. 37C, aligned with the 62/114 structure (residues 54 to 122 ofSEQ ID NO:72). Several different approaches were used to link thecontact sequences, including direct linkage without a spacer (shown as agap in oligonucleotide 62-114b; SEQ ID NO:105), several differentdinucleotides, as shown (62-114a [SEQ ID NO:106]; 62-114c [SEQ IDNO:107]; 62-114d [SEQ ID NO:108]), or d-spacers (62-114e [SEQ IDNO:109]) (Glen Research Corp. (Sterling, Va.)), indicated as “D”s, usingone D for each spacer group (i.e., DD indicates two such spacers used insequence).

The efficacy of these probes in binding the folded target is showngraphically at the bottom of FIG. 37C. The letters below each barindicate the identity of the space, with “NS” indicating no spacer. Thecapture reactions were performed as described above, and the numbers atthe left of the panel indicate the fluorescence measured from thecaptured target DNA/probe complex, shown as a percentage of the signalmeasured when the same amplicons capture a linear (nonbridging) controloligonucleotide 5′-FL TCC TTG ATC GCG G-3′ (SEQ ID NO:123). It can beseen from these data that a combination of CFLP®, computer foldmodeling, and PCR walking can be used to successfully design probescapable of binding to non-contiguous sites on the target molecule.Bridge probes having the “TT” spacer and mismatches to the target withineither contact sequence, similar to those demonstrated in the bridgeprobes in Example 7, show very little binding to the rpoB DNA (signalequal to no-target background; data not shown), confirming thatinteraction of both contact sequences is necessary.

In selection of a probe to span this structure, some spacers show betterperformance than others. While the binding performance of the probes inFIG. 37C is well above background, it is possible that a differentspacer might enhance binding without changing the contact sequences.Similarly, different spacers may perform differently in the enzymaticreactions described in Examples 9-11. If finding the optimal spacer isdesired for any given application of these bridging probes, a morecomprehensive comparison may be performed. For example, a simple, yetbroad test would be to assess all possible dinucleotide arrangements, 16possibilities in all, in addition to the no spacer and non-nucleotidespacer options. While other lengths of contact sequence may be used, theuse of contact sequences of eight nucleotides on either side of the stemis convenient for a first test and gives a reasonable probability ofsuccess. If desired, shorter contact sequences may be tried, either inthe first test or after an optimal spacer arrangement has beenidentified. Given the ease and low cost of current methods of automatedoligonucleotide synthesis, the creation of this number of test probeswould not be burdensome.

Similar approaches were used in the design of bridging probes to otherpredicted structures within the rpoB amplicon. Some of these structuresare shown schematically in FIGS. 38A, 38B, and 38C. For comparison, the62-114 structure with oligonucleotide 62-114 (a) (SEQ ID NO:106) isreproduced in FIG. 38C. In each of these figures the base pair analyzedby CFLP®, PCR walking, and folding predictions is at the base of thedepicted stem, and the nucleotide positions measured from the 5′ end ofthe DNA fragment are indicated by arrows. The corresponding bridgingprobes (53-118(cg) [SEQ ID NO:110]; 69-110(cg) [SEQ ID NO:111];75-121(a)(ta) [SEQ ID NO:112]; 75-121(b)(ta) [SEQ ID NO:113]; 78-106(cg)[SEQ ID NO:114]; 63-87(gc) [SEQ ID NO:115]; 84-102(at) [SEQ ID NO:116];57-119(at) [SEQ ID NO:117]; 62-113 [SEQ ID NO:118]; and 62-98 [SEQ IDNO:119]) are identified by these same basepair numbers (e.g., the probedesigned to span the basepair formed between nucleotides 75 and 121 istermed 75-121). If two probes were targeted to the same basepair theprobes are further distinguished by lower case letters (e.g., 75-121(a)and 75-121(b)). In the case of the 75-121 probes, the target materialdid not have a full 8 nucleotides 3′ of the base of the structure, so abridging probe having only 7 nucleotides at this position was created(75-121(a); SEQ ID NO:112). Because PCR products may include anon-templated “A” nucleotide at the 3′ ends (shown in parentheses), abridging probe have an extra “T” nucleotide was created. The presence ofthis basepair would extend this contact sequence duplex to 8nucleotides. All probes were designed with two 8 nucleotide contactsequences (complementary to the target) flanking a 2 nucleotide spacer.Each of these three figures includes a graph of the fluorescence signalmeasured after the solid support capture of each amplicon by theindicated probe. The numbers identifying the probes used in each capturetest are indicated below each bar. The signal is shown as a percentageof the signal detected by binding of a linear (non-bridging) fullycomplementary probe. While some of these probes have poor bindingproperties (i.e., less than about 5% of the signal from the linearcontrol oligonucleotide), these data further demonstrate the efficacy ofthis method at identifying non-contiguous target sequences that can bebound by a single bridging probe.

As noted above, it is possible for several different structuralconformers to contribute a single band in a CFLP® cleavage pattern. Thismeans that the nucleotide upstream of the cleavage site can pair withseveral different downstream nucleotides at different times, or ondifferent copies of the nucleic acid molecule in a population. RecallingPCR walking data from the investigation of the pairing partners fornucleotide 62 and 63 in the rpoB amplicon, shown in FIG. 37B, it wasseen that there was residual cleavage at position 62 even when thepreferred structure was disrupted by deletion and mutation in theamplicon. This indicates that there might be other, less favored foldedconformations contributing to cleavage at this site. One way of lookingfor such alternative conformations is to carefully examine the lessenergetically favored structures predicted by a program such as mfold.Such analysis was done to identify other regions to which nucleotides 62and 63 might pair. The primary 62/114 structure and two less favorablevariants are shown schematically in FIG. 39. Bridging probes weredesigned to test the for the presence of each of these variantstructures. These are shown schematically in FIGS. 40-42.

It was recognized that representation of these alternative structures inthe molecule populations, as measured by bridge probe binding, waslikely to be influenced by the length of the target molecule by any oneof a number of mechanisms, including but not limited to the following:longer molecules may have a more diverse population of possiblestructures, making any one sub-optimal structure a lower percentage ofthe signal; the additional sequences present may provide regions ofcomplementarity that compete with the some portion of the less favoredstructure, thereby reducing its presence in the population; additionalsequences may form additional stems that do not interact directly withthe less favored structure, but that nonetheless inhibit probe bindingby steric or other interactions. To investigate this effect the bridgesdesigned to bind to the structures depicted in FIG. 39 were tested usingtarget molecules of several lengths. The full length (i.e., the 128-mer)amplicon (SEQ ID NO:72) allows the most favored structure shown in FIG.39(a) to form, and allows a full 8 nucleotides of contact with probe62-114 on either side of the structure. Deletion of the target to 121nucleotides (SEQ ID NO:76) reduces the downstream contact of the 62-114probe to 7 nucleotides, yet allows a full 8 nucleotides of hybridizationfor the 62-113 probe designed to bind to variant 39(b). Binding of aprobe to this structure would create a four way “Holliday” junction.Even though nucleotides 62 and 113 are not basepaired in this structure,this nomenclature is used for the probes oligonucleotide to reflect thepositions of the contact sequences within the target strand. To exploreeven less favored structures, the target was further truncated to 113nucleotides, eliminating regions complementary to both the 62-114 and62-113 probes. The substitution of a C for the wild-type G at position113 (“113 MM”, SEQ ID NO:92) causes mismatches in the basepairing ofnucleotide 113 in both structures 39(a) and 39(b), although withdifferent putative pairing partners.

Each of FIGS. 40, 41, and 42 includes a graph of the fluorescence signalmeasured after the solid support capture of each amplicon by theindicated probe. The numbers identifying the version of the targetmolecule used in each capture test are indicated below each bar. Thesignal is shown as a percentage of the signal detected by binding of alinear (non-bridging) fully complementary probe.

The capture data in FIG. 4 suggests that a structure bridging probe canbe made to cross the base of a sequence capable of forming 2 hairpins.The increase in signal observed when the 121 nucleotide amplicon istargeted suggests that this truncation increases the percentage of thepopulation that is adopting this conformation. The shorted variant, 113MM, was not tested with this probe because one of the two contact siteson the target is deleted in this variant, so binding would not beexpected.

A bridging probe designed to cross only one of the two stems ofconformation 39(b) was also designed (62-98, SEQ ID NO:119), and isshown schematically in FIG. 41. With this probe the presence of thesecond, shorter stem in this conformation would be expected to weaken orblock binding. The target variant having the “C” nucleotide at position113 would have a less stable, shorter stem and would be expected to showmore binding to this probe. The capture data with this probedemonstrates that the majority of the full length amplicon assumes astructure that does not allow binding of this probe. When the target isshortened to 121, more of the molecules fold, such that these bindingsequences are accessible. Finally, when the molecule is shortened to 113nucleotides and the alternative conformations are destabilized, thebinding signal from the 62-98 bridging probe is over 80% of the signalfrom the non-bridging control, verifying that the percentage of themolecular population adopting this previously sub-optimal conformationhas dramatically increased.

Another sub-optimal conformer is predicted in addition to that depictedin FIG. 41. This other variant is shown schematically in FIG. 42, andpredicts basepairing between nucleotide 63 and nucleotide 87. Binding ofthe 63-87 probe (SEQ ID NO:115) follows a profile similar to thatobserved with the 62-98 probe; this structure does not appear to form ina significant population of either the 128-mer or 121-mer targetmolecules. When the target is both shortened, and the 113 “C” mutationis added, the binding at this site is markedly increased, yielding asignal about 13% of that from the non-bridging control. It is notsurprising that it does not increase to the same extent as the 62-98structure, because it represents an alternative conformer of the samemolecule (the 113 MM target) and, absent any conformational shiftactually promoted by the binding of the probe, the presence of the 62-98structure would block binding of this probe.

These data clearly show that distal sequences can have an effect onlocal structures, which is consistent with earlier observations (Brow,et al. supra). The structure analysis method of the present inventionprovides a way of clearly identifying the regions of structuralinteraction. However, it is envisioned that this method has utilitybeyond the design and optimization of bridging probes. This type ofstructure analysis can also be used to improve the performance of otheranalysis methods based on structure. For example, some regions of genesare refractory to CFLP” and/or SSCP analysis because the mutations donot detectably alter the conformations of the folded target nucleicacids. In other applications a sites on a molecule that would be usefulfor hybridization (e.g., for detection, analysis, or antisense purposes)might be inaccessible due to strand folding. The knowledge gained inusing the structure analysis method described herein allows selection oftarget materials or sites more amenable to these methods. For example,PCR primers used to generate the materials for the CFLP® and SSCPanalysis may be relocated to eliminate undesirable structuralinteractions, or they may include mutations or extra sequences chosen tospecifically alter the folding behavior of the material. PCR primersmight include a region of complementarity to a selected part of theresulting amplicon strand, the sequestration of which would cause a siteof interest to be disposed in a more desirable conformation (i.e., morerevealing of mutation or polymorphism, or more accessible tohybridization for other purposes). In another embodiment, undesirablestructures may be disrupted by the provision of an additionalhybridization probe. Clearly, such disrupting probes need not interactdirectly with, or adjacent to the site of interest; it is envisionedthat binding of such disrupting probes may be at a far removed locationfrom the site of interest. The only requirement is that the binding ofthe probe cause a favorable change in the conformation assumed by thenucleic acid of interest. Such effect may be fairly direct (e.g., bydirect blocking of the formation of an undesirable structure) or may beindirect (e.g., by precipitating a chain of conformational shifts thatultimately result in the elimination of an undesirable structure). Thislatter embodiment, in which the disrupter sequence is not made to be apart of the same strand as the sequence of interest, would haveparticular application in antisense applications in vivo.

EXAMPLE 14 Bridging Oligonucleotides

Using the structure analysis methods described above, new bridgingoligonucleotides were designed for the target HCV 244bp DNA, which isthe same target used before. One set of probes was designed to span astructure predicted to form with a base pair between 161 and 205 (FIG.43A) (residues 136 to 213 of SEQ ID NO:124), while the other wasdesigned to span a newly identified structure formed with the base pairbetween 33 and 77 (FIG. 43B) (residues 22 to 125 of SEQ ID NO:125).

Three bridging oligonucleotides, shown as G161/C205(7), G33/C77 (7) andG33/C77 (8) (SEQ ID NOS:120,121, and 122, respectively), were used, andthese had 7 or 8 nucleotides of complementarity, respectively, to eachside of hairpins formed in the HCV targets, subtypes 1a, 1b, 2a/c , and3a (SEQ ID NOS:26-29). They were synthetically labeled with fluoresceinat their 5′ ends and purified by gel-electrophoresis. A hybridizationmixture was assembled containing 10-20 fmols of a biotin-labeled testHCV amplicon, (prepared as described in Example 3, but using thebiotinylated primer described in Example 8) 1.5 pmole of one of thefluorescein-labeled capture probes, 0.01 mg/ml tRNA and 0.2% acetylatedBSA, in 150 μl of 4.5× SSPE. The mixture was incubated at roomtemperature for 30 minutes.

Aliquots (100 μl) of the mixtures were then transferred to wells in astreptavidin-coated 96-well plate (Boehringer Mannheim) and incubated atroom temperature for 20 minutes. The plate was then washed three timeswith TBS (25 mM Tris-Cl, 0.15 M NaCl, pH 7.2) with 0.01% Tween®-20non-ionic detergent. Then, 100 μl of a 1:5000 dilution of 0.75 μl/mianti-fluorescein antibody conjugated with alkaline-phosphatase in 0.2%I-block buffer (Tropix, Bedford, Mass.) was added to each well. After 20minutes at room temperature, the plate was washed three times with TBSwith 0.01% Tween®-20. Then, 100 μl of Attophos fluorescent substrate(JBL, San Louis Obisbo, Calif.) were added to each well and the platewas incubated at 37° C. for 1 hour, before fluorescence readings weretaken using a Perkin-Elmer Cytofluor4000 set to excite at 450/50 nm andto and detect emission at 580/50 nm. Each assay was performed induplicate with the standard deviation represented by the black bar atthe top of each column in the FIGS. 44A and 44B, the fluorescenceintensity is indicated in arbitrary fluorescence units.

These data show that the use of shorter contact sequences can enhancethe discriminating power of the structure probing of variants usingbridge probes. The data from capture by the G33/C77 (8) probe (SEQ IDNO:122), shown in FIG. 44A, can be compared to the center panel of FIG.44B, which shows the signals from the G33/C77 (7) probe (SEQ ID NO:121).The latter probe binds the same structure as the former, but has only 7nt of complementarity on either side of the spacer. Even though thetotal fluorescence signal is reduced, the use of shorter probe resultsin a greater difference in signal between the different HCV genotypes,allowing more accurate identification of these types. Similarly, the useof the G161/C205 (7) probe (SEQ ID NO:120), which is similar to probe“b” (SEQ ID NO:53) described in Example 8 but is one nt shorter oneither terminus, shows the same effect. Examination of the binding of“b” to the same four types of HCV, shown in FIGS. 19 and 25 demonstratesthat types 1a, 1b and 2a/c produce similar amounts of signal compared tothe non-bridging control shown in each panel; 3a does not efficientlybind probe “b”. In comparison, the capture signals from the shorterprobe G161/C205 (7), shown in the right hand panel of FIG. 44B show muchgreater discrimination between the 1a, 1b and 2a/c normalized signals,each being distinct from the others. These data demonstrate that the useof probes having shorter contact sequences can allow more sensitivedistinction between the structures assumed by closely related nucleicacid molecules (i.e., those differing in sequence by only one or a fewnucleotides).

It is also clear from the above that the present invention providesmethods for the analysis of secondary structure within nucleic acids,without the need for either electrophoretic separation of conformationsor fragments or for elaborate and expensive methods of visualizing gels(e.g., darkroom supplies, blotting equipment or fluorescence imagers).The novel methods of the present invention allow the rapididentification of variants (e.g., mutations) within genes obtained fromvarious organisms, including humans.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

128 1 391 DNA Mycobacterium tuberculosis 1 agctcgtatg gcaccggaaccggtaaggac gcgatcacca gcggcatcga ggtcgtatgg 60 acgaacaccc cgacgaaatgggacaacagt ttcctcgaga tcctgtacgg ctacgagtgg 120 gagctgacga agagccctgctggcgcttgg caatacaccg ccaaggacgg cgccggtgcc 180 ggcaccatcc cggacccgttcggcgggcca gggcgctccc cgacgatgct ggccactgac 240 ctctcgctgc gggtggatccgatctatgag cggatcacgc gtcgctggct ggaacacccc 300 gaggaattgg ccgacgagttcgccaaggcc tggtacaagc tgatccaccg agacatgggt 360 cccgttgcga gataccttgggccggtggtc c 391 2 391 DNA Mycobacterium tuberculosis 2 agctcgtatggcaccggaac cggtaaggac gcgatcacca ccggcatcga ggtcgtatgg 60 acgaacaccccgacgaaatg ggacaacagt ttcctcgaga tcctgtacgg ctacgagtgg 120 gagctgacgaagagccctgc tggcgcttgg caatacaccg ccaaggacgg cgccggtgcc 180 ggcaccatcccggacccgtt cggcgggcca gggcgctccc cgacgatgct ggccactgac 240 ctctcgctgcgggtggatcc gatctatgag cggatcacgc gtcgctggct ggaacacccc 300 gaggaattggccgacgagtt cgccaaggcc tggtacaagc tgatccaccg agacatgggt 360 cccgttgcgagataccttgg gccgctggtc c 391 3 391 DNA Mycobacterium tuberculosis 3agctcgtatg gcaccggaac cggtaaggac gcgatcacca gcggcatcga ggtcgtatgg 60acgaacaccc cgacgaaatg ggacaacagt ttcctcgaga tcctgtacgg ctacgagtgg 120gagctgacga agagccctgc tggcgcttgg caatacaccg ccaaggacgg cgccggtgcc 180ggcaccatcc cggacccgtt cggcgggcca gggcgctccc cgacgatgct ggccactgac 240ctctcgctgc gggtggatcc gatctatgag cggatcacgc gtcgctggct ggaacacccc 300gaggaattgg ccgacgagtt cgccaaggcc tggtacaagc tgatccaccg agacatgggt 360cccgttgcga gataccttgg gccgctggtc c 391 4 391 DNA Mycobacteriumtuberculosis 4 agctcgtatg gcaccggaac cggtaaggac gcgatcacca ccggcatcgaggtcgtatgg 60 acgaacaccc cgacgaaatg ggacaacagt ttcctcgaga tcctgtacggctacgagtgg 120 gagctgacga agagccctgc tggcgcttgg caatacaccg ccaaggacggcgccggtgcc 180 ggcaccatcc cggacccgtt cggcgggcca gggcgctccc cgacgatgctggccactgac 240 ctctcgctgc gggtggatcc gatctatgag cggatcacgc gtcgctggctggaacacccc 300 gaggaattgg ccgacgagtt cgccaaggcc tggtacaagc tgatccaccgagacatgggt 360 cccgttgcga gataccttgg gccggtggtc c 391 5 20 DNAArtificial Sequence Synthetic 5 agctcgtatg gcaccggaac 20 6 20 DNAArtificial Sequence Synthetic 6 ttgacctccc acccgacttg 20 7 21 DNAArtificial Sequence Synthetic 7 agctcgtatg gcaccggaac c 21 8 20 DNAArtificial Sequence Synthetic 8 ggaccagcgg cccaaggtat 20 9 22 DNAArtificial Sequence Synthetic 9 ggaccaccgg cccaaggtat ct 22 10 21 DNAArtificial Sequence Synthetic 10 tttttgccgc tggtgatcgc g 21 11 12 DNAArtificial Sequence Synthetic 11 ggagagccat ag 12 12 11 DNA ArtificialSequence Synthetic 12 tggtctgcgg a 11 13 11 DNA Artificial SequenceSynthetic 13 ggacgaccgg g 11 14 11 DNA Artificial Sequence Synthetic 14ggagatttgg g 11 15 11 DNA Artificial Sequence Synthetic 15 ccgcgagact g11 16 12 DNA Artificial Sequence Synthetic 16 ctagccgagt ag 12 17 11 DNAArtificial Sequence Synthetic 17 tgttgggtcg c 11 18 11 DNA ArtificialSequence Synthetic 18 ccgcgagacc g 11 19 11 DNA Artificial SequenceSynthetic 19 ccgcaagacc g 11 20 289 DNA Hepatitis C virus 20 gattctgtcttcacgcagaa agcgtctagc catggcgtta gtatgagtgt cgtgcagcct 60 ccaggaccccccctcccggg agagccatag tggtctgcgg aaccggtgag tacaccggaa 120 ttgccaggacgaccgggtcc tttcttggat caacccgctc aatgcctgga gatttgggcg 180 tgcccccgcaagactgctag ccgagtagtg ttgggtcgcg aaaggccttg tggtactgcc 240 tgatagggtgcttgcgagtg ccccgggagg tctcgtagac cgtgcaatc 289 21 286 DNA Hepatitis Cvirus 21 gattctgtct tcacgcagaa agcgtctagc catggcgtta gtatgagtgtcgtgcagcct 60 ccaggtcccc ccctcccggg agagccatag tggtctgcgg aaccggtgagtacaccggaa 120 ttgccaggac gaccgggtcc tttcttggat caacccgctc aatgcctggagatttgggcg 180 tgcccccgcg agactgctag ccgagtagtg ttgggtcgcg aaaggccttgtggtactgcc 240 tgatagggtg cttgcgagtg ccccgggagg tctcgtagac cgtgca 286 22289 DNA Hepatitis C virus 22 gattctgtct tcacgcagaa agcgtctagc catggcgttagtatgagtgt cgtacagcct 60 ccaggccccc ccctcccggg agagccatag tggtctgcggaaccggtgag tacaccggaa 120 ttgccgggaa gactgggtcc tttcttggat aaacccactctatgcccggc catttgggcg 180 tgcccccgca agactgctag ccgagtagcg ttgggttgcgaaaggccttg tggtactgcc 240 tgatagggtg cttgcgagta ccccgggagg tctcgtagaccgtgcaatc 289 23 289 DNA Artificial Sequence Synthetic 23 gattctgtcttcacgcagaa agcgcctagc catggcgtta gtacgagtgt cgtgcagcct 60 ccaggaccccccctcccggg agaaccatag tggtctgcgg aaccggtgag tacaccggaa 120 tcgctggggtgaccgggtcc tttcttggag caacccgctc aatacccaga aatttgggcg 180 tgcccccgcgagatcactag ccgagtagtg ttgggtcgcg aaaggccttg tggtactgcc 240 tgatagggtgcttgcgagtg ccccgggagg tctcgtagac cgtgcaatc 289 24 18 DNA ArtificialSequence Synthetic 24 ctcgcaagca ccctatca 18 25 21 DNA ArtificialSequence Synthetic 25 gcagaaagcg tctagccatg g 21 26 244 DNA Hepatitis Cvirus 26 gcagaaagcg tctagccatg gcgttagtat gagtgtcgtg cagcctccaggaccccccct 60 cccgggagag ccatagtggt ctgcggaacc ggtgagtaca ccggaattgccaggacgacc 120 gggtcctttc ttggatcaac ccgctcaatg cctggagatt tgggcgtgcccccgcaagac 180 tgctagccga gtagtgttgg gtcgcgaaag gccttgtggt actgcctgatagggtgcttg 240 cgag 244 27 244 DNA Hepatitis C virus 27 gcagaaagcgtctagccatg gcgttagtat gagtgtcgtg cagcctccag gtccccccct 60 cccgggagagccatagtggt ctgcggaacc ggtgagtaca ccggaattgc caggacgacc 120 gggtcctttcttggatcaac ccgctcaatg cctggagatt tgggcgtgcc cccgcgagac 180 tgctagccgagtagtgttgg gtcgcgaaag gccttgtggt actgcctgat agggtgcttg 240 cgag 244 28244 DNA Hepatitis C virus 28 gcagaaagcg tctagccatg gcgttagtat gagtgtcgtacagcctccag gcccccccct 60 cccgggagag ccatagtggt ctgcggaacc ggtgagtacaccggaattgc cgggaagact 120 gggtcctttc ttggataaac ccactctatg cccggccatttgggcgtgcc cccgcaagac 180 tgctagccga gtagcgttgg gttgcgaaag gccttgtggtactgcctgat agggtgcttg 240 cgag 244 29 244 DNA Hepatitis C virus 29gcagaaagcg cctagccatg gcgttagtac gagtgtcgtg cagcctccag gaccccccct 60cccgggagaa ccatagtggt ctgcggaacc ggtgagtaca ccggaatcgc tggggtgacc 120gggtcctttc ttggagcaac ccgctcaata cccagaaatt tgggcgtgcc cccgcgagat 180cactagccga gtagtgttgg gtcgcgaaag gccttgtggt actgcctgat agggtgcttg 240cgag 244 30 216 DNA Hepatitis C virus 30 cagaaagggt ttagccatggggttagtatg agtgtcgtac agcctccagg cccccccctc 60 ccgggagagc catagtggtctgcggaaccg gtgagtacac cggaattgcc gggaagactg 120 ggtcctttct tggataaacccactctatgc ccggccattt gggcgtgccc ccgcaagact 180 gctagccgag tagcgttgggttgcgaaagg ccttgt 216 31 244 DNA Hepatitis C virus 31 cagaaagggtttagccatgg cgttagtatg agtgtcgtgc agcctccagg accccccctc 60 ccgggagagccatagtggtc tgcggaaccg gtgagtacac cggaattgcc aggacgaccg 120 ggtcctttcttggataaaac ccgctcaatg cctggagatt tgggcgtgcc cccgcaagac 180 tgctagccgagtagtgttgg gtcgcgaaag gccttgtggt actgcctgat agggtgcttg 240 caag 244 32239 DNA Hepatitis C virus 32 gcagaaaggt ttagccatgg gttagtatga gtgtcgtgcagcctccagga ccccccctcc 60 cgggagagcc atagtggtct gcggaaccgg tgagtacaccggaattgcca ggacgaccgg 120 gtcctttctt ggattaaccc gctcaatgcc tggagatttgggcgtgcccc cgcaagactg 180 ctagccgagt agtgttgggt cgcgaaaggc cttgtggtactgcctgatag ggtgcttgc 239 33 240 DNA Hepatitis C virus 33 gcagaaaggtttagccatgg ggttagtatg agtgtcgtac agcctccagg accccccctc 60 ccgggagagccatagtggtc tgcggaaccg gtgagtacac cggaattgcc aggacgaccg 120 ggtcctttcttggataaacc cgctcaatgc ctggagattt gggcgtgccc ccgcaagact 180 gctagccgagtagtgttggg tcgcgaaagg ccttgtggta ctgcctgata gggtgcttgc 240 34 240 DNAHepatitis C virus 34 gcagaaaggg tttagccatg gcgttagtat gagtgtcgtacagcctccag gcccccccct 60 cccgggagag ccatagtggt ctgcggaacc ggtgagtacaccggaattac cggaaagact 120 gggtcctttc ttggataaac ccactctatg tccggtcatttgggcgtgcc cccgcaagac 180 tgctagccga gtagcgttgg gttgcaaagg ccttgtggtactgcctgata gggtgcttgc 240 35 240 DNA Hepatitis C virus 35 cagaaagggtttagccatgg ggttagtacg agtgtcgtgc agcctccagg cccccccctc 60 ccgggagagccatagtggtc tgcggaaccg gtgagtacac cggaatcgct ggggtgaccg 120 ggtcctttcttggagcaacc cgctcaatac ccagaaattt gggcgtgccc ccgcgagatc 180 actagccgagtagtgttggg tcgcgaaagg ccttgtggta ctgcctgata gggtgcttgc 240 36 239 DNAHepatitis C virus 36 agaaagcgtt tagccatggc gttagtatga gtgttgtgcagcctccagga ccccccctcc 60 cgggagagcc atagtggtct gcggaaccgg tgagtacaccggaattgcca ggacgaccgg 120 gtcctttctt ggatcaaccc gctcaatgcc tggagatttgggcgtgcccc cgcaagactg 180 ctagccgagt agtgttgggt cgcgaaaggc cttgtggtactgcctgatag ggtgcttgc 239 37 232 DNA Hepatitis C virus 37 gtttagccatggcgttagta tgagtgtcgt gcagcctcca ggaccccccc tcccgggaga 60 gccatagtggtctgcggaac cggtgagtac accggaattg ccaggacgac cgggtccttt 120 cttggatcaacccgctcaat gcctggagat ttgggcgtgc ccccgcgaga ccgctagccg 180 agtagtgttgggtcgcgaaa ggccttgtgg tactgcctga tagggtgctt gc 232 38 240 DNA HepatitisC virus 38 gcagaaagcg tttagccatg gcgttagtac gagtgtcgtg cagcctccaggaccccccct 60 cccgggagag ccatagtggt ctgcggaacc ggtgagtaca ccggaatcgctggggtgacc 120 gggtcctttc ttggaacaac ccgctcaata cccagaaatt tgggcgtgcccccgcgagat 180 cactagccga gtagtgttgg gtcgcgaaag gccttgtggt actgcctgatagggtgcttg 240 39 44 DNA Artificial Sequence Synthetic 39 tgctctctggtcgctgtctg aaagacagcg tggtctctcg taat 44 40 44 DNA Artificial SequenceSynthetic 40 tgctctctgg tcgctgtctg aaagactccg tggtctctcg taat 44 41 44DNA Artificial Sequence Synthetic 41 tgctctctgg tcgctgtctg aatttttttttggtctctcg taat 44 42 14 DNA Artificial Sequence Synthetic 42 agaccattaccaga 14 43 16 DNA Artificial Sequence Synthetic 43 gagaccatta ccagag 1644 18 DNA Artificial Sequence Synthetic 44 agagaccatt accagaga 18 45 18DNA Artificial Sequence Synthetic 45 agagaccatt acaagcga 18 46 18 DNAArtificial Sequence Synthetic 46 agcgaacatt accagaga 18 47 16 DNAArtificial Sequence Synthetic 47 agagaccaac cagaga 16 48 9 DNAArtificial Sequence Synthetic 48 agagaccat 9 49 9 DNA ArtificialSequence Synthetic 49 taccagaga 9 50 10 DNA Artificial SequenceSynthetic 50 accagagagc 10 51 10 DNA Artificial Sequence Synthetic 51tcagacagcg 10 52 18 DNA Artificial Sequence Synthetic 52 agtggtctgcggaaccgg 18 53 18 DNA Artificial Sequence Synthetic 53 agtgtcgtttggaaccgg 18 54 18 DNA Artificial Sequence Synthetic 54 agtgtcgtaaggaaccgg 18 55 18 DNA Artificial Sequence Synthetic 55 agtgtcgtcaggaaccgg 18 56 16 DNA Artificial Sequence Synthetic 56 agtgtcgtgg aaccgg16 57 18 DNA Artificial Sequence Synthetic 57 agtgtcgttt ggatccgg 18 5818 DNA Artificial Sequence Synthetic 58 agtgacgttt ggaaccgg 18 59 8 DNAArtificial Sequence Synthetic 59 ggaaccgg 8 60 20 DNA ArtificialSequence Synthetic 60 ttttgtgagt acaccggaat 20 61 14 DNA ArtificialSequence Synthetic 61 ttttgtgagt acac 14 62 15 DNA Artificial SequenceSynthetic 62 tgagtacacc ggaat 15 63 33 DNA Artificial Sequence Synthetic63 attccggtgt actcaccggt tccaaacgac act 33 64 18 DNA Artificial SequenceSynthetic 64 cagcctcccc ttcttgga 18 65 20 DNA Artificial SequenceSynthetic 65 agtgtcgttt ggaattaatt 20 66 16 DNA Artificial SequenceSynthetic 66 gcgaaaggcc ttgtgg 16 67 16 DNA Artificial SequenceSynthetic 67 acagcctcca ggaccc 16 68 16 DNA Artificial SequenceSynthetic 68 gcagcctcca ggaccc 16 69 193 DNA Mycobacterium tuberculosis69 cgtggaggcg atcacaccgc agacgttgat caacatccgg ccggtggtcg ccgcgatcaa 60ggagttcttc ggcaccagcc agctgagcca attcatggac cagaacaacc cgctgtcggg 120gttgacccac aagcgccgac tgtcggcgct ggggcccggc ggtctgtcac gtgagcgtgc 180cgggctggag gtc 193 70 26 DNA Artificial Sequence Synthetic 70 cgtggaggcgatcacaccgc agacgt 26 71 25 DNA Artificial Sequence Synthetic 71gacctccagc ccggcacgct cacgt 25 72 128 DNA Mycobacterium tuberculosis 72cgccgcgatc aaggagttct tcggcaccag ccagctgagc caattcatgg accagaacaa 60cccgctgtcg gggttgaccc acaagcgccg actgtcggcg ctggggcccg gcggtctgtc 120acgtgagc 128 73 20 DNA Artificial Sequence Synthetic 73 cgccgcgatcaaggagttct 20 74 20 DNA Artificial Sequence Synthetic 74 gctcacgtgacagaccgccg 20 75 18 DNA Artificial Sequence Synthetic 75 tgacagaccgccgggccc 18 76 121 DNA Artificial Sequence Synthetic 76 cgccgcgatcaaggagttct tcggcaccag ccagctgagc caattcatgg accagaacaa 60 cccgctgtcggggttgaccc acaagcgccg actgtcggcg ctggggcccg gcggtctgtc 120 a 121 77 18DNA Artificial Sequence Synthetic 77 agacagaccg ccgggccc 18 78 121 DNAArtificial Sequence Synthetic 78 cgccgcgatc aaggagttct tcggcaccagccagctgagc caattcatgg accagaacaa 60 cccgctgtcg gggttgaccc acaagcgccgactgtcggcg ctggggcccg gcggtctgtc 120 t 121 79 18 DNA Artificial SequenceSynthetic 79 acagaccgcc gggcccca 18 80 119 DNA Artificial SequenceSynthetic 80 cgccgcgatc aaggagttct tcggcaccag ccagctgagc caattcatggaccagaacaa 60 cccgctgtcg gggttgaccc acaagcgccg actgtcggcg ctggggcccggcggtctgt 119 81 18 DNA Artificial Sequence Synthetic 81 ccagaccgccgggcccca 18 82 119 DNA Artificial Sequence Synthetic 82 cgccgcgatcaaggagttct tcggcaccag ccagctgagc caattcatgg accagaacaa 60 cccgctgtcggggttgaccc acaagcgccg actgtcggcg ctggggcccg gcggtctgg 119 83 18 DNAArtificial Sequence Synthetic 83 cagaccgccg ggccccag 18 84 118 DNAArtificial Sequence Synthetic 84 cgccgcgatc aaggagttct tcggcaccagccagctgagc caattcatgg accagaacaa 60 cccgctgtcg gggttgaccc acaagcgccgactgtcggcg ctggggcccg gcggtctg 118 85 18 DNA Artificial SequenceSynthetic 85 gagaccgccg ggccccag 18 86 118 DNA Artificial SequenceSynthetic 86 cgccgcgatc aaggagttct tcggcaccag ccagctgagc caattcatggaccagaacaa 60 cccgctgtcg gggttgaccc acaagcgccg actgtcggcg ctggggcccggcggtctc 118 87 20 DNA Artificial Sequence Synthetic 87 ccgccgggccccagcgccga 20 88 114 DNA Artificial Sequence Synthetic 88 cgccgcgatcaaggagttct tcggcaccag ccagctgagc caattcatgg accagaacaa 60 cccgctgtcggggttgaccc acaagcgccg actgtcggcg ctggggcccg gcgg 114 89 20 DNAArtificial Sequence Synthetic 89 gcgccgggcc ccagcgccga 20 90 114 DNAArtificial Sequence Synthetic 90 cgccgcgatc aaggagttct tcggcaccagccagctgagc caattcatgg accagaacaa 60 cccgctgtcg gggttgaccc acaagcgccgactgtcggcg ctggggcccg gcgc 114 91 20 DNA Artificial Sequence Synthetic91 cggccgggcc ccagcgccga 20 92 114 DNA Artificial Sequence Synthetic 92cgccgcgatc aaggagttct tcggcaccag ccagctgagc caattcatgg accagaacaa 60cccgctgtcg gggttgaccc acaagcgccg actgtcggcg ctggggcccg gccg 114 93 18DNA Artificial Sequence Synthetic 93 cgggccccag cgccgaca 18 94 110 DNAArtificial Sequence Synthetic 94 cgccgcgatc aaggagttct tcggcaccagccagctgagc caattcatgg accagaacaa 60 cccgctgtcg gggttgaccc acaagcgccgactgtcggcg ctggggcccg 110 95 18 DNA Artificial Sequence Synthetic 95agggccccag cgccgaca 18 96 110 DNA Artificial Sequence Synthetic 96cgccgcgatc aaggagttct tcggcaccag ccagctgagc caattcatgg accagaacaa 60cccgctgtcg gggttgaccc acaagcgccg actgtcggcg ctggggccct 110 97 18 DNAArtificial Sequence Synthetic 97 ccccagcgcc gacagtcg 18 98 106 DNAArtificial Sequence Synthetic 98 cgccgcgatc aaggagttct tcggcaccagccagctgagc caattcatgg accagaacaa 60 cccgctgtcg gggttgaccc acaagcgccgactgtcggcg ctgggg 106 99 18 DNA Artificial Sequence Synthetic 99tcccagcgcc gacagtcg 18 100 106 DNA Artificial Sequence Synthetic 100cgccgcgatc aaggagttct tcggcaccag ccagctgagc caattcatgg accagaacaa 60cccgctgtcg gggttgaccc acaagcgccg actgtcggcg ctggga 106 101 20 DNAArtificial Sequence Synthetic 101 cgcttgtggg tcaaccccga 20 102 87 DNAArtificial Sequence Synthetic 102 cgccgcgatc aaggagttct tcggcaccagccagctgagc caattcatgg accagaacaa 60 cccgctgtcg gggttgaccc acaagcg 87 10320 DNA Artificial Sequence Synthetic 103 agcttgtggg tcaaccccga 20 104 87DNA Artificial Sequence Synthetic 104 cgccgcgatc aaggagttct tcggcaccagccagctgagc caattcatgg accagaacaa 60 cccgctgtcg gggttgaccc acaagct 87 10516 DNA Artificial Sequence Synthetic 105 gtgacagagt tgttct 16 106 18 DNAArtificial Sequence Synthetic 106 gtgacagatt gttgttct 18 107 18 DNAArtificial Sequence Synthetic 107 gtgacagagc gttgttct 18 108 18 DNAArtificial Sequence Synthetic 108 gtgacagaaa gttgttct 18 109 16 DNAArtificial Sequence Synthetic 109 gtgacagagt tgttct 16 110 18 DNAArtificial Sequence Synthetic 110 tcacgtgagc gtccatga 18 111 18 DNAArtificial Sequence Synthetic 111 cagaccgcgc acagcggg 18 112 17 DNAArtificial Sequence Synthetic 112 gctcacgata ccccgac 17 113 18 DNAArtificial Sequence Synthetic 113 tgctcacgat accccgac 18 114 18 DNAArtificial Sequence Synthetic 114 cgccgggcgc tcaacccc 18 115 18 DNAArtificial Sequence Synthetic 115 acagtcgggc ggttgttc 18 116 18 DNAArtificial Sequence Synthetic 116 cgggccccta tgtgggtc 18 117 18 DNAArtificial Sequence Synthetic 117 ctcacgtgta tctggtcc 18 118 16 DNAArtificial Sequence Synthetic 118 tgacagacgt tgttct 16 119 18 DNAArtificial Sequence Synthetic 119 ccccagcggc gttgttct 18 120 16 DNAArtificial Sequence Synthetic 120 gtgtcgtttg gaaccg 16 121 16 DNAArtificial Sequence Synthetic 121 tgggcgttgc ttgtgg 16 122 18 DNAArtificial Sequence Synthetic 122 ttgggcgttg cttgtggt 18 123 13 DNAArtificial Sequence Synthetic 123 tccttgatcg cgg 13 124 244 DNAHepatitis C virus 124 ctcgcaagca ccctatcagg cagtaccaca aggcctttcgcgacccaaca ctactcggct 60 agcagtcttg cgggggcacg cccaaatctc caggcattgagcgggttgat ccaagaaagg 120 acccggtcgt cctggcaatt ccggtgtact caccggttccgcagaccact atggctctcc 180 cgggaggggg ggtcctggag gctgcacgac actcatactaacgccatggc tagacgcttt 240 ctgc 244 125 244 DNA Hepatitis C virus 125ctcgcaagca ccctatcagg cagtaccaca aggcctttcg cgacccaaca ctactcggct 60agcagtctcg cgggggcacg cccaaatctc caggcattga gcgggttgat ccaagaaagg 120acccggtcgt cctggcaatt ccggtgtact caccggttcc gcagaccact atggctctcc 180cgggaggggg ggacctggag gctgcacgac actcatacta acgccatggc tagacgcttt 240ctgc 244 126 244 DNA Hepatitis C virus 126 ctcgcaagca ccctatcaggcagtaccaca aggcctttcg caacccaacg ctactcggct 60 agcagtcttg cgggggcacgcccaaatggc cgggcataga gtgggtttat ccaagaaagg 120 acccagtctt cccggcaattccggtgtact caccggttcc gcagaccact atggctctcc 180 cgggaggggg gggcctggaggctgtacgac actcatacta acgccatggc tagacgcttt 240 ctgc 244 127 244 DNAHepatitis C virus 127 ctcgcaagca ccctatcagg cagtaccaca aggcctttcgcgacccaaca ctactcggct 60 agtgatctcg cgggggcacg cccaaatttc tgggtattgagcgggttgct ccaagaaagg 120 acccggtcac cccagcgatt ccggtgtact caccggttccgcagaccact atggttctcc 180 cgggaggggg ggtcctggag gctgcacgac actcgtactaacgccatggc taggcgcttt 240 ctgc 244 128 244 DNA Hepatitis C virus 128cucgcaagca cccuaucagg caguaccaca aggccuuucg cgacccaaca cuacucggcu 60agcagucuug cgggggcacg cccaaaucuc caggcauuga gcggguugau ccaagaaagg 120acccggucgu ccuggcaauu ccgguguacu caccgguucc gcagaccacu auggcucucc 180cgggaggggg gguccuggag gcugcacgac acucauacua acgccauggc uagacgcuuu 240cugc 244

We claim:
 1. A method, comprising: a) providing: i) target nucleic acidcomprising first and second non-contiguous single-stranded regionsseparated by an intervening region comprising a double-stranded region,wherein said target nucleic comprises at least a portion of Hepatitis Cvirus nucleic acid; ii) a bridging oligonucleotide capable of binding tosaid first and second non-contiguous single-stranded regions; iii) asecond oligonucleotide capable of binding to a portion of said firstnon-contiguous single-stranded region; and iii) a cleavage agent; b)mixing said target nucleic acid, said bridging oligonucleotide, saidsecond oligonucleotide, and said cleavage agent under conditions suchthat a cleavage structure is formed from said target nucleic acid, saidbridging oligonucleotide, and said second oligonucleotide, and whereineither said second oligonucleotide or said bridging oligonucleotide iscleaved by said cleavage agent.
 2. The method of claim 1, wherein saidcleavage agent comprises a nuclease.
 3. The method of claim 2, whereinsaid cleavage agent comprises a thermostable 5′ nuclease.
 4. The methodof claim 3, wherein said thermostable 5′ nuclease comprises a modifiedpolymerase; wherein said modified polymerase is a modified nativepolymerase of Thermus species.
 5. The method of claim 2, wherein saidnuclease is selected from the group consisting of Pyrococcus woesiiPEN-1 endonuclease, Methanococcus jannaschii FEN-1 endonuclease,Pyrococcus furiosus FEN-1 endonuclease, and Archaeoglobus fulgidus FEN-1endonuclease.
 6. The method of claim 1, wherein said conditions of saidmixing allow for hybridization of said bridging oligonucleotide and saidsecond oligonucleotide to said target nucleic acid so as to define aregion of overlap of said oligonucleotides.
 7. The method of claim 6,wherein said region of overlap comprises one base.
 8. The method ofclaim 6, wherein said region of overlap comprises more than one base. 9.The method of claim 1, wherein said Hepatitis C virus is selected fromthe group consisting of Hepatitis C virus variants 1a, 1b, 2a/c , and3a.
 10. A method, comprising: a) providing: i) target nucleic acidcomprising first and second non-contiguous single-stranded regionsseparated by an intervening region, said intervening region comprising afirst double-stranded portion and a second double-stranded portionseparated by a connecting single-stranded portion, wherein said targetnucleic acid comprises at least a portion of Hepatitis C virus nucleicacid; and ii) a bridging oligonucleotide capable of binding to saidfirst and second non-contiguous single-stranded regions; and b) mixingsaid target nucleic acid and said bridging oligonucleotide underconditions such that said bridging oligonucleotide hybridizes to saidtarget to form an oligonucleotide/target complex.
 11. The method ofclaim 10, wherein said Hepatitis C virus is selected from the groupconsisting of Hepatitis C virus variants 1a, 1b, 2a/c, and 3a.
 12. Amethod, comprising: a) providing: i) target nucleic acid comprisingfirst and second non-contiguous single-stranded regions separated by anintervening region comprising a double-stranded portion, wherein saidtarget nucleic acid comprises at least a portion of Hepatitis C virusnucleic acid; ii) a bridging oligonucleotide capable of binding to saidfirst and second non-contiguous single-stranded regions; and iii) areactant selected from the group consisting of polymerases and ligases;and b) mixing said target nucleic acid, said bridging oligonucleotideand said reactant under conditions such that said bridgingoligonucleotide hybridizes to said target nucleic acid, and wherein saidbridging oligonucleotide is modified by said reactant to produce amodified oligonucleotide.
 13. The method of claim 12, wherein saidreactant is a polymerase, and said modified oligonucleotide comprises anextended oligonucleotide.
 14. The method of claim 12, wherein saidreactant is a ligase, and said modified oligonucleotide comprises aligated oligonucleotide.
 15. The method of claim 12, wherein saidbridging oligonucleotide is capable of binding to fewer than tennucleotides of each of said first and second non-contiguoussingle-stranded regions.
 16. The method of claim 15, wherein saidbridging oligonucleotide is capable of binding to seven or fewernucleotides of each of said first and second non-contiguoussingle-stranded regions.
 17. The method of claim 12, wherein saidHepatitis C virus is selected from the group consisting of Hepatitis Cvirus variants 1a, 1b, 2a/c , and 3a.
 18. A method for detecting thepresence of a target nucleic acid molecule by detecting non-targetcleavage products comprising: a) providing: i) a cleavage agent; ii)Hepatitis C virus target nucleic acid, said target nucleic acidcomprising a first region and a second region, said second regiondownstream of and contiguous to said first region; iii) a firstoligonucleotide, wherein at least a portion of said firstoligonucleotide is completely complementary to said first portion ofsaid first target nucleic acid; iv) a second oligonucleotide comprisinga 3′ portion and a 5′ portion, wherein said 5′ portion is completelycomplementary to said second portion of said target nucleic acid; b)mixing said cleavage agent, said target nucleic acid, said firstoligonucleotide and said second oligonucleotide to create a reactionmixture under reaction conditions such that at least said portion ofsaid first oligonucleotide is annealed to said first region of saidtarget nucleic acid and wherein at least said 5′ portion of said secondoligonucleotide is annealed to said second region of said target nucleicacid so as to create a cleavage structure, and wherein cleavage of saidcleavage structure occurs to generate non-target cleavage product; andc) detecting the cleavage of said cleavage structure.
 19. The method ofclaim 18, wherein said detecting the cleavage of said cleavage structurecomprises detecting said non-target cleavage product.
 20. The method ofclaim 18, wherein said 3′ portion of said second oligonucleotidecomprises a 3′ terminal nucleotide not complementary to said targetnucleic acid.
 21. The method of claim 18, wherein said 3′ portion ofsaid second oligonucleotide consists of a single nucleotide notcomplementary to said target nucleic acid.
 22. The method of claim 18,wherein either said first oligonucleotide or said second oligonucleotidecomprises a fluorescent label and said detecting the cleavage of saidcleavage structure comprises detection of fluorescence from saidfluorescent label.
 23. The method of claim 18, wherein said detectingthe cleavage of said cleavage structure comprises detection of mass. 24.The method of claim 18, wherein said first and second oligonucleotidescollectively comprise a fluorescence energy donor and a fluorescenceenergy acceptor and wherein said detecting the cleavage of said cleavagestructure comprises detection of fluorescence energy transfer betweensaid fluorescence energy donor and said fluorescence energy acceptor.25. The method of claim 18, wherein said either said firstoligonucleotide or said second oligonucleotide comprises a labelselected from the group consisting of a radioactive label, a luminescentlabel, a phosphorescent label, a fluorescence polarization label, andcharge label and detecting the cleavage of said cleavage structurecomprises detection selected from the group consisting of detection ofradioactivity, luminescence, phosphorescence, fluorescence polarization,and charge from said label.
 26. The method of claim 18, wherein saidfirst oligonucleotide is attached to a solid support.
 27. The method ofclaim 18, wherein said second oligonucleotide is attached to a solidsupport.
 28. The method of claim 18, wherein said cleavage agentcomprises a structure-specific nuclease.
 29. The method of claim 28,wherein said structure-specific nuclease comprises a thermostablestructure-specific nuclease.
 30. The method of claim 29, wherein saidcleavage agent comprises a 5′ nuclease.
 31. The method of claim 31,wherein said 5′-nuclease comprises a thermostable 5′-nuclease.
 32. Themethod of claim 31, wherein a portion of the amino acid sequence of saidnuclease is homologous to a portion of the amino acid sequence of athermostable DNA polymerase from a thermophilic organism.
 33. The methodof claim 32, wherein said thermophilic organism is selected from thegroup consisting of Thermus aquaticus, Thermus flavus, and Thermusthermophilus.
 34. The method of claim 18, wherein said detecting thecleavage of said cleavage structure comprises: a) providing: i) saidnon-target cleavage product; ii) a composition comprising twosingle-stranded nucleic acids annealed so as to define a single-strandedportion of a protein binding region; and iii) a protein; and b)contacting said non-target cleavage product and said single-strandedportion of said protein binding region under conditions such that saidnon-target cleavage product and said single-stranded portion of aprotein binding region hybridize to form a double stranded proteinbinding region, and wherein said protein binds to said double strandedprotein binding region.
 35. The method of claim 34, wherein said proteincomprises a nucleic acid polymerase and wherein said nucleic acidpolymerase binds to said protein binding region and produces nucleicacid.
 36. The method of claim 35, wherein said protein binding region isa template-dependent RNA polymerase binding region.
 37. The method ofclaim 36, wherein said template-dependent RNA polymerase binding regionis a T7 RNA polymerase binding region.
 38. The method of claim 18,wherein said detecting the cleavage of said cleavage structurecomprises: a) providing: i) said non-target cleavage product; ii) asingle continuous strand of nucleic acid comprising a sequence defininga single strand of an RNA polymerase binding region; iii) atemplate-dependent DNA polymerase; and iv) a template-dependent RNApolymerase; b) contacting said non-target cleavage product and said RNApolymerase binding region under conditions such that said non-targetcleavage product binds to a portion of said single strand of said RNApolymerase binding region to produce a bound non-target cleavageproduct; c) contacting said bound non-target cleavage product and saidtemplate-dependent DNA polymerase under conditions such that adouble-stranded RNA polymerase binding region is produced; and d)contacting said double-stranded RNA polymerase binding region and saidtemplate-dependent RNA polymerase under conditions such that RNAtranscripts are produced.
 39. The method of claim 38, further comprisingthe step of e) detecting said RNA transcripts.
 40. The method of claim38, wherein said template-dependent RNA polymerase is T7 RNA polymerase.41. The method of claim 18, wherein said target nucleic acid comprisessingle-stranded DNA.
 42. The method of claim 18, wherein said targetnucleic acid comprises double-stranded DNA and prior to step c), saidreaction mixture is treated such that said double-stranded DNA isrendered substantially single-stranded.
 43. The method of claim 42,wherein said double-stranded DNA is rendered substantiallysingle-stranded by heat.
 44. The method of claim 18, wherein saidreaction conditions comprise providing a source of divalent cations. 45.The method of claim 44, wherein said divalent cation is selected fromthe group consisting of Mn²⁺ and Mg²⁺ ions.
 46. The method of claim 18,wherein said first and said second oligonucleotides are provided inconcentration excess compared to said target nucleic acid.
 47. Themethod of claim 18, further comprising providing a third oligonucleotidecomplementary to a third portion of said target nucleic acid upstream ofsaid first portion of said first target nucleic acid, wherein said thirdoligonucleotide is mixed with said reaction mixture in step b); andwherein said third oligonucleotide is annealed to said third portion ofsaid target nucleic acid.